Methods of diagnosing and treating cd55 deficiency, hyperactivation of complement, angiopathic thrombosis and protein losing enteropathy (chaple), a newly identified orphan disease

ABSTRACT

Disclosed herein are methods of diagnosing, and treating and/or preventing CD55-deficiency, hyperactivation of complement, angiopathic thrombosis and protein-losing enteropathy (CHAPLE). The method of diagnosing includes: providing a sample from a patient; performing an assay detecting at least one of at least one mutation in a DNA sequence of a CD55 gene, at least one mutation in a RNA sequence of a CD55 transcript, at least one mutation in a DNA sequence of a CD55 complementary-DNA (cDNA), CD55 protein, CD55 protein binding, complement deposition or combinations thereof; and diagnosing the patient with CHAPLE. The method of treating and/or preventing at least one symptom of CHAPLE includes: administering an effective amount of a composition comprising at least one complement inhibitor to a subject in need thereof, wherein the composition is effective in treating or preventing at least one symptom of CHAPLE. The disclosure further relates to compositions effective at treating and/or preventing CHAPLE.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/394,630, filed on 14 Sep. 2016, the contents of which areincorporated here by reference in its entirety.

GOVERNMENT FUNDING

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services.

INCORPORATION BY REFERENCE

In compliance with 37 C.F.R. § 1.52(e)(5), the sequence informationcontained in electronic file name:1420378_459WO2_Sequence_Listing_ST25.txt; size 77 KB; created on: 24Aug. 2017; using Patent-In 3.5, and Checker 4.4.0 is hereby incorporatedherein by reference in its entirety.

BACKGROUND 1. Field of the Invention

The present disclosure relates to methods of detecting mutationsassociated with and diagnosing CD55 deficiency, hyperactivation ofcomplement, angiopathic thrombosis and protein losing enteropathy(CHAPLE), methods for the treatment of CHAPLE, and therapeuticcompositions for treating CHAPLE.

2. Background Invention

A rare disease is generally accepted as being any disease that affects asmall percentage of the population, but there is no single definition.For example, rare diseases are referred to as Orphan Diseases in theUnited States and defined as conditions that affect fewer than 200,000people in the United States. Orphan diseases include, inter alia, cysticfibrosis, Lou Gehrig's disease, and Tourette's syndrome, Hamburgerdisease, Job syndrome, atypical hemolytic uremic syndrome, paroxysmalnocturnal hemoglobinuria, acromegaly (or “gigantism”). An accuratediagnosis of a rare disease can generally take up to 5 years becauseearly stage symptoms could be absent, masked, misunderstood, or confusedwith other more prevalent diseases. New orphan diseases are discoveredeach year and are typically caused by inherited gene mutations.

Protein-losing enteropathy (PLE) is characterized by excessive loss ofserum proteins through the gastrointestinal (GI) tract resulting inhypoproteinemia, edema, and, in some cases, pleural and pericardialeffusions. Pathogenic mechanisms of PLE can involve (1) impaired barrierfunction of the GI mucosa, often in association with inflammatory boweldisease (IBD) and (2) impaired lymphatic drainage, either due to primaryintestinal lymphangiectasia, or secondary to systemic conditions thatimpair lymph flow, such as cardiac diseases. Although most cases of PLEare sporadic, primary intestinal lymphangiectasia has been reported inmultiple siblings of several families, suggesting a genetic etiology incertain cases.

Identifying rare monogenetic defects can elucidate the pathophysiologyof disease, improve diagnosis, and promote targeted therapies forspecific inherited syndromes and related common diseases. These effortshave been greatly aided by massively parallel DNA sequencingtechnologies, increasingly comprehensive datasets of human geneticvariation, and new gene validation technologies. Several intestinaldiseases related to early onset IBD (EO-IBD) have been attributed toMendelian gene defects. Recently, a loss-of-function (LOF) mutation inthe plasmalemma vesicle associated protein gene was reported in a younginfant with severe PLE associated with disruption of endothelialfenestrated diaphragms in the intestinal vasculature, demonstrating thatPLE may also arise from Mendelian gene defects.

The complement system is a vital part of the immune system of the bodythat protect against pathogens (e.g., bacteria, fungi, viruses, etc.).The complement system is a complex collection of greater than 25 plasmaproteins and membrane factors. The complement components interactthrough a series of intricate enzymatic cleavages and membrane bindingevents, which result in the production of products with opsonic,immunoregulatory, and lytic functions. Because complement has potenteffects on immunity and cell physiology, it must be tightly controlled.

The complement cascade includes three pathways referred to as theclassical pathway, the lectin pathway, or the alternative pathway. Theclassical pathway of complement activation is initiated or triggered byan antibody recognizing (i.e., binding) an antigen located on a targetcell. The lectin pathway of complement activation is initiated ortriggered by the binding of a mannose-binding protein that is present inblood plasma to mannose-containing proteoglycans on the surfaces ofbacteria and yeast, which has structural similarities to the antibodyinitiation of the classical pathway. As such, the lectin pathway thenproceeds in a similar fashion as the classical pathway. The alternativepathway of complement activation is initiated or triggered byconstituents of bacterial surfaces, as will be discussed in greaterdetail below.

Each of the three pathways lead to the covalent bonding of a particularfragment of a complement component (i.e., the C3b fragment of C3) to thepathogen surface, which is recognized by C3b receptors on macrophagesand neutrophils. This deposition of C3b fragments on the surface of thepathogen is mediated by a C3 convertase, which is a protease thatcleaves the complement component C3 to yield C3a and C3b. The C3bfragment is an osponin, which mediates interaction with phagocytes(e.g., macrophages, neutrophils, and dendritic cells, which promoteinflammatory immune reactions) through their C3b receptors. As such, theopsonized pathogens are targeted by phagocytes. The opsonic function ofC3b is one of the most important functions of the complement system, andits disruption leads to a susceptibility to a broad range of pathogens.C3b can then be progressively broken down by Factor I (a protease) and acofactor (i.e., Factor H, CR1, MCP, or C4BP) to iC3b, then C3c+C3dg, andfinally C3d. The C3a fragment is a potent anaphylatoxin that stimulatesmast cell degranulation, which results in the release of histamine frombasophils and mast cells. Histamine enhances vascular permeability,smooth muscle contraction, and leukocyte activation, as well as being anattractant (chemotractic factor) for granulocytes (e.g., neutrophils,eosinophils, basophils) and macrophages.

Complement component C3, which is the most abundant complement proteinand is abundantly present in plasma, is spontaneously hydrolyzed (thealternative pathway). In particular, there is a spontaneous cleavage ofa thioester bond in C3 that forms C3i or C3(H₂O). The alternativepathway is facilitated by surfaces that support the binding of activatedC3 (i.e., C3i or C3(H₂O)), as well as surfaces that have neutral orpositive charge characteristics (such as those found in bacteria). Theplasma protein Factor B binds to the C3i surface bound protein. Factor Dthen subsequently cleaves Factor B, thereby producing Ba and Bb. The Bbfragment remains bound to C3i to form a C3iBb, which is a C3 convertasethat functions as described above and may be referred to as thealternative pathway C3 convertase. The alternative pathway C3 convertaseis stabilized by the binding of properdin (although not required). Thealternative pathway C3 convertase has an amplification affect becauseeach of the fluid-phase C3 convertase (i.e., the alternative pathway C3convertase) can cleave multiple C3 proteins into C3a and C3b, whichresults in the deposition of additional C3b covalently bound to thesurface (e.g., a bacterial surface). In a similar fashion, thealternative pathway C5 convertase is formed by the addition of a secondC3b monomer to the alternative pathway C3 convertase (i.e., add C3b toC3iBb, which is referred to as (C3b)₂Bb), which is also stabilized bythe binding of properdin (although not required). The alternativepathway C3 convertase bind and cleaves C5.

The classical pathway C3 convertase is formed when complement componentC1 (comprising a complex of C1q, C1r, and C1s) is activated by anantibody-target-antigen complex (e.g., a microbial antigen). That is,the binding of C1q to the antibody-target-antigen complex results in aconformational change in C1, which activates C1r. Activated C1rcleaves/activates C1s. Active C1s is a serene protease that cleavescomplement component C4 into C4b and C4a and complement component C2into C2b and C2a. C4b fragments contain a reactive thiol that readilyforms amide or ester bonds with suitable molecules on a target surface(e.g., a microbial cell surface). Activated C4b and C2a form theclassical pathway C3 convertase, which functions as described above.When a C3b monomer is added to the classical pathway convertase, theclassical pathway C5 convertase (C4b, C2a, C3b) is formed.

As discussed above, the lectin pathway is homologous to the classicalpathway with a mannose-binding lectin (MBL, which is similar to C1q) tomannose residues on the surface of a pathogen. This binding activatesthe MBL-associated serine proteases MASP-1 and MASP-2 (which are verysimilar to C1r and C1s, respectively). This complex functions in thesame fashion as C1 in the classical complement pathway.

The alternative, classical, and lectin pathway C5 convertases cleave C5,which is found in normal human serum, into C5a and C5b. Similar to C3a,C5a is a potent anaphylatoxin and chemotactic factor. C5a receptors arepresent on the surfaces of: bronchial and alveolar epithelial cells,bronchial smooth muscle cells, eosinophils, mast cells, monocytes,neutrophils, and activated lymphocytes.

C5b binds to C6 and C7 to form a complex that interacts with C8 andsubsequently numerous C9 proteins, thereby producing a membrane attackcomplex (MAC) or lytic unit (C5b,6,7,8)₁(9)_(n)(n=10-16 molecules) onthe targeted cell membrane, which is a pore that extends through thecell membrane. When a sufficient number of MACs are present in thetarget cell membranes, hypotonic lysis of the targeted cell is achieved.C5a and C5b-9 also amplify the release of downstream inflammatoryfactors (e.g., hydrolytic enzymes, reactive oxygen species, arachidonicacid metabolites and various cytokines).

The complement system provides a robust defense against infection.However, misregulation or inappropriate activation of the complementsystem is associated with the pathogenesis of a variety of disordersincluding (e.g., rheumatoid arthritis; lupus nephritis; asthma;ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS);dense deposit disease; paroxysmal nocturnal hemoglobinuria (PNH);macular degeneration; hemolysis, elevated liver enzymes, and lowplatelets syndrome; thrombotic thrombocytopenic purpura; spontaneousfetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrentfetal loss; multiple sclerosis; traumatic brain injury; and injuryresulting from myocardial infarction, cardiopulmonary bypass andhemodialysis). Complement inhibition has been demonstrated to beeffective in treating several complement-associated disorders in animalmodels and in humans (see, e.g., Rother et al. (2007) NatureBiotechnology 25(11):1256-1264; Wang et al. (1996) Proc Natl Acad SciUSA 93:8563-8568; Wang et al. (1995) Proc Natl Acad Sci USA92:8955-8959; Rinder et al. (1995) J Clin Invest 96:1564-1572; Kroshuset al. (1995) Transplantation 60:1194-1202; Homeister et al. (1993) JImmunol 150:1055-1064; Weisman et al. (1990) Science 249:146-151;Amsterdam et al. (1995) Am J Physiol 268:H448-H457; and Rabinovici etal. (1992) J Immunol 149:1744 1750.

CD55 (decay accelerating factor or DAF) is a widely expressed GPI-linkedcell surface protein that regulates complement activation by reducingthe activity of C3 and C5 convertases and accelerating theirdisassembly. Specifically, DAF recognizes C4b and C3b fragments that arecreated during the C4 activation (classical complement pathway andlectin pathway) and C3 activation (alternate complement pathway). DAFinterferes with the conversion of C2 to C2a through its interaction withthe cell-associated C4b of the classical and lectin pathways. Thisprevents the formation of the C4b2a C3 convertase. Similarly, DAFinterferes with the conversion of factor B to Bb by factor D through itsinteraction with C3b of the alternative pathway. This prevents theformation of the C3bBb C3 convertase of the alternative pathway. As aresult, the amplification provided by 3C convertases of the complementcascade are limited by the interaction of DAF (or CD55) with C4b andC3b, which results in indirectly blocking the formation of the MAC. Arare CD55 deficiency on red blood cells (RBCs), known as the Inabphenotype, is detected by the loss of Cromer blood group antigens. Thisrare CD55 deficiency has been associated with complement dysregulation,formation of a strong RBC alloantibody agglutinin, and GI abnormalities.However, a disease resulting from the germline loss of CD55 has not beenclearly defined to date.

As mentioned above, rare diseases are often misdiagnosed, and new rarediseases are regularly identified. For example, the present disclosuredescribes a newly identified rare disease—CHAPLE disease/syndrome. Assuch, there exists a need to accurately detect the mutations associatedwith CHAPLE disease/syndrome, as well as diagnose and treat individualswith CHAPLE disease/syndrome with a therapeutic composition.

SUMMARY

The present disclosure relates to the surprising and unexpecteddiscovery of the cause of CHAPLE, and therefore, methods of diagnosingCHAPLE, as well as methods of treating patients with CHAPLE symptom(s)and methods of preventing a symptom(s) of CHAPLE in individualspredisposed to develop CHAPLE syndrome with a therapeutic composition.

In an aspect, the disclosure provides a method of diagnosing a patientwith CD55 deficiency, hyperactivation of complement, angiopathicthrombosis and protein losing enteropathy (CHAPLE). The methodcomprises: providing a sample from a patient; performing an assay todetect at least one of: at least one mutation in a DNA sequence of aCD55 gene, at least one mutation in a RNA sequence (or mRNA sequence) ofa CD55 transcript, at least one mutation in a DNA sequence of a CD55complementary DNA (cDNA), decay-accelerating factor (DAF) or CD55protein, complement deposition, or a combination thereof, and diagnosingthe patient with CHAPLE.

In an embodiment, the patient is diagnosed with CHAPLE when at least oneof the following is detected: (i) at least one mutation in a DNAsequence, an RNA sequence or a cDNA sequence of CD55 that results in aCD55 protein with substantially diminished functional activity, a CD55protein with no functional activity, a lack of expression of CD55protein (i.e., no CD55 protein expression), or a substantiallydiminished expression of CD55 protein; (ii) a CD55 protein withsubstantially diminished functional activity; (iii) a CD55 protein withno functional activity; (iv) a lack of expression of CD55 protein (i.e.,no CD55 protein expression); (v) a substantially diminished expressionof CD55 protein; or (vi) a combination thereof.

In another embodiment, the patient is diagnosed with CHAPLE when thepatient has at least one CHAPLE related symptom and at least one of thefollowing is detected: (i) at least one mutation in a DNA sequence, anRNA sequence or a cDNA sequence of CD55 that results in a CD55 proteinwith substantially diminished functional activity, a CD55 protein withno functional activity, a lack of expression of CD55 protein (i.e., noCD55 protein expression), or a substantially diminished expression ofCD55 protein; (ii) a CD55 protein with substantially diminishedfunctional activity; (iii) a CD55 protein with no functional activity;(iv) a lack of expression of CD55 protein (i.e., no CD55 proteinexpression); (v) a substantially diminished expression of CD55 protein;or (vi) a combination thereof.

In some embodiments, the CHAPLE related symptom is selected from thegroup consisting of: inflammatory bowel disease, enteropathy, proteinlosing enteropathy, protein losing enteropathy associated withhypoalbuminemia, hypoalbuminemia, hypogammaglobulinemia, intestinallymphangiectasia, lymphangiectasia, thrombotic events, thromboembolism,hyperactivation of complement, angiopathic thrombosis, hypoproteinemia,or a combination thereof.

In other embodiments, the CHAPLE related symptom is selected from thegroup consisting of inflammatory bowel disease, protein losingenteropathy associated with hypoalbuminemia, hypogammaglobulinemia,intestinal lymphangiectasia, thrombotic events or a combination thereof.For example, in certain embodiments, the patient has at least 3 of thefollowing symptoms: inflammatory bowel disease, protein losingenteropathy associated with hypoalbuminemia, hypogammaglobulinemia,intestinal lymphangiectasia, or thrombotic events.

In a particular embodiment, the method further comprises administeringan effective mount of a composition comprising at least one complementinhibitor to the subject with CHAPLE, wherein the composition iseffective in treating or preventing at least one symptom of CHAPLE.

In a further embodiment, the complement inhibitor is selected from thegroup consisting of a serine protease inhibitor, a soluble complementregulator, a therapeutic antibody or an antigen-binding fragmentthereof, a complement component inhibitor, and an anaphylatoxin receptorantagonist.

In certain embodiments, the mutation in the DNA sequence of the CD55gene or in the RNA sequence of the CD55 transcript results in a near tocomplete absence of CD55 protein expression or the expression of CD55protein with substantially diminished function or that isnon-functional.

In an embodiment, the mutation in the DNA sequence of the CD55 gene isat least one of c.149-150delAA, c.149-150insCCTT, c.109delC, c.800G>C,c.287-1G>C, c.149-150delAAinsCCTT or a combination thereof.

In further embodiments, detecting includes at least one of: (i)sequencing at least a portion of the CD55 gene or the CD55 transcript orthe CD55 cDNA; or (ii) contacting a labeled nucleic acid probe to atleast a portion of the CD55 gene or the CD55 transcript or the CD55cDNA; or (iii) contacting at least a portion of the CD55 gene or CD55transcripts or the CD55 cDNA with a microarray; or (iv) a combinationthereof.

In a particular embodiment, hybridization of the labeled nucleic acidprobe is indicative of a mutation in the portion of the CD55 gene or theCD55 transcript or the CD55 cDNA. In another particular embodiment,hybridization of the labeled nucleic acid probe is indicative of theportion of the CD55 gene or the CD55 transcript or the CD55 cDNA havinga wild-type sequence at the location of hybridization.

In certain other embodiments, sequencing at least a portion of the CD55gene or CD55 transcript or CD55 cDNA thereof includes amplifying atleast one region of interest for sequencing with at least one polymerasechain reaction (PCR) that includes at least one of the following primersets: (i) CTACTCCACCCGTCTTGTTTGT and TTTGGGGGTTAAGGATACAGTC (Exon 1);(ii) CAGGTGTGGCATTTCAAGG and ACCCTGGGGTTTAGTAACGC (Exon 2); (iii)AAGTACTAAATATGCGCAAAGCAG and ATGGTCCTATCAAGAAACATCC (Exon 3); (iv)GTTACCTTCTTTGTGTGTATGCC and GCTGTGAATACCAGTCATGAAAC (Exon 4); (v)AACCTGGAGAATTTGAGGAAAG and TGTGCTAATATTCTTAAGGGGC (Exon 5); (vi)GCATTTATAAGCATCTCTTGTTGG and TCATTGAATGTCTGCAACCC (Exon 6); (vii)CTAGGTGTTTGTGGGGAGAGAG and TCTGGTGGGTTTCTGAAGAGTT (Exon 7); (viii)TTTACGCAGAGTCCTTCAGC and CCATTTAATCCTGCAATCTTGG (Exon 8); (ix)TGGAAATTTGAGTTGCTTTCG and TCTCCCAGGAATATGGATTG (Exon 9); (x)GCACCCCAAATTAACTGATTC and ATGTGATTCCAGGACTGCC (Exon 10); or (xi) acombination thereof.

In yet another embodiment, contacting a labeled nucleic acid probe to atleast a portion of the CD55 gene or the CD55 transcript or the CD55 cDNAis performed using real-time PCR. In an embodiment, hybridization of areal-time PCR probe is indicative of a mutation in the portion of theCD55 gene or CD55 transcript or the CD55 cDNA. In another embodiment,hybridization of a real-time PCR probe is indicative of the portion ofthe CD55 gene or CD55 transcript or the CD55 cDNA having a wild-typesequence (or not having a CHAPLE related mutations) at the location ofhybridization.

In some embodiments, contacting a labeled nucleic acid probe to at leasta portion of the CD55 cDNA comprises: isolating CD55 transcripts;reverse transcribing at least a portion of the CD55 transcripts; andcontacting the cDNA with the labeled nucleic acid probe.

In further embodiments, the microarray includes probes (e.g.,immobilized probes) designed to detect DNA, transcripts (i.e., RNA ormRNA), or cDNA mutations that result in the complete absence in CD55protein or a CD55 protein with substantially diminished function or thatis virtually non-functional.

In certain embodiments, detecting CD55 protein comprises: contacting thesample with at least one CD55 binding polypeptide. The CD55 bindingpolypeptides can include a detectable label. Furthermore, the bindingpolypeptide can be an anti-CD55 antibody or a CD55-binding fragmentthereof.

In other embodiments, detecting CD55 protein further comprisescontacting the sample or a CD55-CD55 binding polypeptide complex with atleast one secondary polypeptide that binds specifically to the CD55binding polypeptide. The secondary polypeptide can include a detectablelabel. The secondary polypeptide can be an antibody or fragment thereofthat binds the CD55 binding polypeptide.

In an embodiment, detecting CD55 protein is performed using at least oneof the following assays: western blot, flow cytometry, an immunoassay ora combination thereof. For example, the immunoassay can be at least oneassay selected from the group consisting of flow cytometry,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, magneticimmunoassay, enzyme-linked immunospot (ELISPOT), and immunofluorescence.

In another embodiment, the detecting of CD55 binding function includesexamining at least one of C3b affinity, C3b avidity, C4b affinity, C4bavidity or a combination thereof.

In a particular embodiment, detecting complement deposition includesdetecting C3d deposition. In an embodiment, detecting complementdeposition is determined by flow cytometry.

In a further aspect, the disclosure provides a method of treating apatient having CD55 deficiency, hyperactivation of complement,angiopathic thrombosis and protein losing enteropathy (CHAPLE) orpreventing CHAPLE in a patient at risk of developing the same. Themethod comprises administering an effective amount of a compositioncomprising at least one complement inhibitor to a subject in needthereof. The composition is effective in treating and/or preventing atleast one symptom of CHAPLE.

In some embodiments, the complement inhibitor is selected from the groupconsisting of a serine protease inhibitor, a soluble complementregulator, a therapeutic antibody or an antigen-binding fragmentthereof, a complement component inhibitor, and an anaphylatoxin receptorantagonist.

In certain embodiments, the serine protease inhibitors is at least oneof a C3 convertase inhibitor, a C5 convertase inhibitor, a C1 inhibitor,a C1r inhibitor, a C1s inhibitor, a C2a inhibitor, a MASP-1 inhibitor, aMASP-2 inhibitor, a factor D inhibitor, a factor B inhibitor, a factor Iinhibitor or a combination thereof. For example, the serine proteaseinhibitor can be at least one of BCX-1470 (BioCryst, Birmingham, Ala.,USA), C1s-INH-248 (Knoll/Abbott, Abbott Park, Ill., USA), compstatin,Cetor® (Sanquin, Amsterdam, Netherlands), Berinert® (CSL Behring, Kingof Prussia, Pa., USA), Cinryze™ (ViroPharma, Exton, Pa., USA), rhC 1INH(Pharming Group N.V., Leiden, Netherlands), Ruconest® (SalixPharmaceuticals, Inc., Raleigh, N.C., USA) or a combination thereof.

In other embodiments, the soluble complement regulator is at least oneof a soluble form of a membrane cofactor protein (MCP or CD46), asoluble form of a decay-accelerating factor (DAF or CD55), a solubleform of a membrane attack complex-inhibitor protein (MAC-IP or CD59), asoluble form of complement receptor 1 (CD35) or a combination thereof.For example, the soluble complement regulator can be at least one ofsCR1 (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sCR1-sL^(ex) (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge, Mass., USA), amembrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or a combinationthereof.

In further embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one polypeptide that binds C3, C3a, C3b, C3convertase, C5, C5a, C5b, C5 convertase, C7, C8, or C9, factor B, factorD, C4, C2, C1, properdin, a functional blocking antibody of ananaphylatoxin or a combination thereof. The binding can inhibitcomplement activation by at least one of blocking association/bindingwith other complement proteins, blocking association/binding withreceptor proteins, blocking serine protease activity or a combinationthereof. For example, the therapeutic antibody or the antigen-bindingfragment thereof can be at least one of eculizumab (Soliris®; AlexionPharmaceuticals Inc., New Haven, Conn., USA), ALXN1007 (AlexionPharmaceuticals Inc., New Haven, Conn., USA), neutrazumab (G2 Therapies,Darlinghurst, NSW, Australia), Pexelizumab (Alexion PharmaceuticalsInc., New Haven, Conn., USA), ofatumumab (Genmab A/S, Copenhagen,Denmark), HuMax-CD38 (Benmab A/S, Copenhagen, Denmark), TNX-558 (Tanox,South San Francisco, Calif., USA), TNX-234 (Tanox, South San Francisco,Calif., USA), TA106 (Taligen, Aurora, Colo., USA), anti-properdin(Novelmed, Cleveland, Ohio, USA) or a combination thereof.

In yet other embodiments, the complement component inhibitor (e.g., asmall molecule) is a peptide, nucleic acids, a synthetic molecule or acombination thereof that disrupts protein functions by steric hindranceor the induction of conformational changes. For example, the complementcomponent inhibitor can be at least one of compstatin, anti-C5 RNAaptamer (ARC1905; Archemix, Cambridge, Mass., USA), or analogs orderivatives thereof, or a combination thereof.

In another embodiment, the anaphylatoxin receptor antagonist is at leastone of a C5aR antagonist, a C5L2 antagonist, a C3a receptor antagonist,a functional blocking antibody of an anaphylatoxin or a combinationthereof. For example, the anaphylatoxin receptor antagonist is at leastone of PMX-53 (PepTech Corp, Bedform, Mass., USA), PMX-205 (PepTechCorp, Bedform, Mass., USA), JPE-1375 (Jerini, Berlin, Germany), JSM-7717(Jerini, Berlin, Germany), rhMBL (Enzon Pharmaceuticals, Cranford, N.J.,USA), NTD 9513727 (Tocris Bioscience, Bristol, United Kingdom) or acombination thereof.

In another aspect, the disclosure provides a composition for treating orpreventing at least one symptom of CD55 deficiency, hyperactivation ofcomplement, angiopathic thrombosis and protein losing enteropathy(CHAPLE) in a subject in need thereof. The therapeutic compositioncomprises an effective amount of two or more agents and apharmaceutically acceptable carrier, wherein at least one of the agentsis a complement inhibitor, wherein the composition is effective intreating or preventing at least one symptom of CHAPLE. In an embodiment,at least two of the agents are a complement inhibitor. In anotherembodiment the effective amount is a synergistically effective amount ofthe agents.

In an additional embodiment, the complement inhibitor is selected fromthe group consisting of a serine protease inhibitor, a solublecomplement regulator, a therapeutic antibody or an antigen-bindingfragment thereof, a complement component inhibitor (e.g., a smallmolecule), and an anaphylatoxin receptor antagonist.

In further embodiments, the complement inhibitors include a C3convertase inhibitor and a C5 convertase inhibitor. In anotherembodiment, the complement inhibitors includes: a soluble form of CD55;and at least one of a C3 convertase inhibitor, a C5 convertase inhibitoror a combination thereof.

In certain embodiments, the serine protease inhibitors is at least oneof a C3 convertase inhibitor, a C5 convertase inhibitor, a C1 inhibitor,a C1r inhibitor, a C1s inhibitor, a C2a inhibitor, a MASP-1 inhibitor, aMASP-2 inhibitor, a factor D inhibitor, a factor B inhibitor, a factor Iinhibitor or a combination thereof.

In a particular embodiment, the serine protease inhibitor is at leastone of BCX-1470 (BioCryst, Birmingham, Ala., USA), C1s-INH-248(Knoll/Abbott, Abbott Park, Ill., USA), compstatin, Cetor® (Sanquin,Amsterdam, Netherlands), Berinert® (CSL Behring, King of Prussia, Pa.,USA), Cinryze™ (ViroPharma, Exton, Pa., USA), rhC1INH (Pharming GroupN.V., Leiden, Netherlands), Ruconest® (Salix Pharmaceuticals, Inc.,Raleigh, N.C., USA) or a combination thereof.

In other embodiments, the soluble complement regulator is at least oneof a soluble form of a membrane cofactor protein (MCP or CD46), asoluble form of a decay-accelerating factor (DAF or CD55), a solubleform of a membrane attack complex-inhibitor protein (MAC-IP or CD59), asoluble form of complement receptor 1 (CD35) or a combination thereof.

In an embodiment, the soluble complement regulator is at least one ofsCR1 (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sCR1-sL^(ex) (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge, Mass., USA), amembrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or a combinationthereof.

In other embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one polypeptide that binds C3, C3a, C3b, C3convertase, C5, C5a, C5b, C5 convertase, C7, C8, or C9, factor B, factorD, C4, C2, C1, properdin, a functional blocking antibody of ananaphylatoxin or a combination thereof, wherein said binding inhibitscomplement activation by, e.g., at least one of blockingassociation/binding with other complement proteins, blockingassociation/binding with receptor proteins, blocking serine proteaseactivity or a combination thereof.

In certain embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one of eculizumab (Soliris®; AlexionPharmaceuticals Inc., New Haven, Conn., USA), ALXN1007 (AlexionPharmaceuticals Inc., New Haven, Conn., USA), neutrazumab (G2 Therapies,Darlinghurst, NSW, Australia), Pexelizumab (Alexion PharmaceuticalsInc., New Haven, Conn., USA), ofatumumab (Genmab A/S, Copenhagen,Denmark), HuMax-CD38 (Benmab A/S, Copenhagen, Denmark), TNX-558 (Tanox,South San Francisco, Calif., USA), TNX-234 (Tanox, South San Francisco,Calif., USA), TA106 (Taligen, Aurora, Colo., USA), anti-properdin(Novelmed, Cleveland, Ohio, USA) or a combination thereof.

In yet other embodiments, the complement component inhibitor is apeptide, nucleic acids, a synthetic molecule or a combination thereofthat disrupts protein functions by steric hindrance or the induction ofconformational changes.

In a particular embodiment, the complement component inhibitor is atleast one of compstatin, anti-C5 RNA aptamer (ARC1905; Archemix,Cambridge, Mass., USA), or analogs or derivatives thereof, or acombination thereof.

In additional embodiments, the anaphylatoxin receptor antagonist is atleast one of a C5aR antagonist, a C5L2 antagonist, a C3a receptorantagonist, a functional blocking antibody of an anaphylatoxin or acombination thereof.

In certain embodiments, the anaphylatoxin receptor antagonist is atleast one of PMX-53 (PepTech Corp, Bedform, Mass., USA), PMX-205(PepTech Corp, Bedform, Mass., USA), JPE-1375 (Jerini, Berlin, Germany),JSM-7717 (Jerini, Berlin, Germany), rhMBL (Enzon Pharmaceuticals,Cranford, N.J., USA) or a combination thereof.

The preceding general areas of utility are given by way of example onlyand are not intended to be limiting on the scope of the presentdisclosure and appended claims. Additional objects and advantages of thepresent invention will be appreciated by one of ordinary skill in theart in light of the instant claims, description, and examples. Forexample, the various aspects and embodiments of the invention may beutilized in numerous combinations, all of which are expresslycontemplated by the present description. These additional objects andadvantages are expressly included within the scope of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating an embodiment of the invention and are not to be construedas limiting the invention.

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H. Clinical presentation of 7families with familial early onset PLE. Pedigrees of seven families withaffected individuals homozygous for alternative allele indicated bysolid symbols, heterozygous individuals indicated by half solid symbols,and affected individuals with an unknown genotype indicated by opensymbols with a slash through it. The double line between parents in eachfamily indicates presence of consanguinity (A). Serum levels ofimmunoglobulin G (IgG, left Y axis) in relation to serum albumin (rightY axis) concentrations as a function of age in years. Age-specific lowercutoff value for IgG is denoted by the red dotted curve, whereas thereference for albumin level is >3.5 g/dl (indicated by hatched line onright Y axis). Each arrow denotes an episode of pneumonia. Presenteddata for Patients 1.1, 2.1, 3.1, and 4.1 (B). Radiological exams showingbowel wall edema/thickening in Patient 6.1. Double arrows indicatediffuse target-water small bowel wall enhancement and single arrowindicates scattered area of dilatation (C). The hematoxylin and eosinstained histological section from the resection specimen from P2.1 showslymphangiectasia (D). Immunohistochemical stains for PROX-1 and D2-40(Podoplanin) (inset). Radiographs showing large occlusive thrombi in theinferior vena cava (IVC) and right atrium (arrows in E, left).Radiograph showing pulmonary embolus and lack of vascular flow in rightpulmonary artery branches (arrows in E, center). Radiograph showingirregular peripheral arteriovenous malformations (ovals in E, right).Colonoscopy photographs of Patient 1.1 showing exudate formation (upper,arrow) and a mucosal ulcer (lower, arrow) in the terminal ileum (F).Histopathology of colon biopsy specimen taken from hematoxylin and eosinsections of ileum of Patient 2.1 demonstrating prominent lymphoidnodules evident within both the mucosa and beneath the muscularismucosa; and immunohistochemistry showing infiltrates of B cells (CD20)and T cells (CD3) within lymphoid nodules (G). Abdominal CT image fromPatient 2.1 with intestinal obstruction showing mucosal thickening ofthe distal ileal segments and luminal narrowing (the lead pipe sign,arrow) (H).

FIGS. 2A, 2B, 2C, and 2D. Nucleotide and amino acid alignments forc.149_150delAAinsCCTT (A), c.109delG (B), c.287-1G>A (C), c.800G>C (D)to wild-type CD55 sequences (CD55 genomic reference sequenceNG_007465.1, SEQ ID NO: 1, CD55 mRNA reference sequence transcriptNM_000574.4, SEQ ID NO: 2; and CD55 protein reference sequenceNP_000565.1, SEQ ID NO: 3).

FIGS. 3A, 3B, 3C, and 3D. Pedigrees and chromatograms for families 1 and7, families 2, 3, and 5, family 4, and family 6 are shown in 3A, 3B, 3C,and 3D respectively. Extended pedigrees for patient families, withaffected individuals homozygous for alternative allele indicated bysolid symbols, heterozygous individuals indicated by half solid symbols,and affected individuals with an unknown genotype indicated by opensymbols with a slash. The double line between parents in each familyindicates presence of consanguinity. Chromatograms showing the specificnucleotide mutations in the CD55 gene in patients relative to thereference sequence. Families are grouped by unique mutation status.

FIG. 4. Show Table 2, which shows clinical and immunologicalcharacteristics of patients with CD 55 deficiency. Symptoms present atfirst clinical presentation are indicated by red text.‡ The patientreceived colchicine for the treatment of suspected FamilialMediterranean fever (FMF). Numbers in boldface indicate values below thenormal range. N/A: Not applicable (P4.2 and P4.3 were not evaluated byendoscopy). IVC: Inferior vena cava.

FIGS. 5A, 5B, and 5C. Albumin levels and weight/height curves. Serumalbumin levels from P5.1 and 5.2 on a weekly basis and from P6.1 overyears. Periodic abrupt rises represent partial albumin restorationfollowing transfusion (A). Height and weight curves for Patients 1.1,2.1, 6.1, and 7.1 relative to the normal range (B and C).Supplementation therapy with vitamins and dietary intervention led to avariable benefit on patients' growth. Whereas Patient 2.1 was within thethird and tenth weight and height percentiles at presentation, shereached the 25^(th) and 50^(th) percentiles for weight and height,respectively. On the other hand Patient 1.1 had only partial recovery,with persistent stunting despite weight gain. Patient 6.1 demonstratedsignificant growth retardation, worsening after age 14 when the patientdeveloped severe thrombosis. Patient 7.1 has a current height for agevalue below 3^(rd) percentile.

FIGS. 6A, 6B, 6C, 6D, and 6E. Mutations in CD55 lead to loss of proteinexpression. Schematic of the complement cascade (A). The identifiedmutations relative to the CD55 protein structure depicting the fourshort consensus repeat (SCR) domains and exon location (B). QRT-PCRdetermination of CD55 mRNA in cycling patient CD4⁺ T cell blasts (C).Flow cytometry histograms of CD55 surface expression on CD4⁺ Tlymphocytes for the patients compared to healthy controls. Gray shadedhistogram shows isotype control and the red histogram denotesexperimental sample (D). Western blot denoting CD55 expression inactivated CD4⁺ T cells (E).

FIGS. 7A and 7B. 3D structure of CD55 showing mutation in Family 4Location of the mutation in Family 4 within the crystal structure ofCD55 (A). Close up of the affected disulfide bond in SCR4 of CD55 (B).

FIG. 8A, 8B, 8C, 8D, 8E. Loss of CD55 leads to spontaneous complementactivation by the alternative pathway. Representative flow cytometryplots of C3d deposition on the surface of CD4⁺ T cell blasts from P1 andP2 (A, left). Pooled analyses of C3d deposition on T cells of five CD55deficient patients after incubation with normal or acidified (pH 6.4)media containing pooled nHS (A, right). Patient CD4⁺ T cells weretransduced with either wild type (WT) CD55 or Thy1.1 expressinglentivirus (B, top). CD55 or Thy1.1 transduced cells were incubated withacidified normal human serum and the deposition of C3d determined andplotted for the CD55 positive and negative fractions or the Thy1.1positive or negative fractions (B, bottom left and right, respectively).CD55 expression in a CD55-deficient Jurkat cell line (C, left). C3ddeposition on CD55 knock out Jurkat T cells treated with acidified nHSfor the indicated amount of time. Knockout cells were a mixture of cellsthat had been deleted by CRISPR technology and cells that had not been.CD55 positive and negative cells were assessed for C3d deposition withinthis mixed population (C, right). C3d deposition on HT29 cells with CD55knocked down using two different shRNAs. Correlation between thegeometric MFI values of C3d and CD55 was assessed on scatter plotgraphs. Samples were color coded as; Red: Sh-1 CD55, blue: Sh-2 CD55,green: Mock. Circles: No TNF-α pretreatment, Squares: pre-treated withTNF-α. Samples treated with acidified serum are illustrated with solidsymbols and corresponding samples treated with control serum arerepresented with open symbols (D). C3d deposition by live/dead stainingfor control and patient T cells (D, left) and quantification of C3ddeposition and annexin-V/live/dead staining (E).

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 9I, 9J, 9K, 9L, 9M, 9N, 90, and9P.

Changes in inflammatory cytokine production by CD55 deficient cellspromote pro-thrombotic changes in endothelial cells. Cytokine secretionin control and patient T cells following restimulation with anti-CD3 for48 hours. Cells were either left untreated or treated with a combinationof C3aR and C5aR1 inhibitors, both at 10 μM final concentration. Eachpatient point represents the average of at least three independentexperiments. Results are for TNFα, IL10, or IFNγ (A, B, and Crespectively). Anaphylatoxin receptor surface expression onproliferating CD4⁺ T cells grown in complete RPMI supplemented with 100u/mL IL2, isotype: filled grey line, sample: solid black line (D). C3aRexpression on proliferating CD4⁺ T cells grown in serum free X-vivo 15media supplemented with 100 u/mL IL-2 w/wo 10 nM ATRA, grey and blackline respectively, isotype: filled grey line (E). IL-10 production incontrol and patient T cells stimulated with anti-CD3 and stimulatoryantibodies directed against the indicated costimulatory molecules (F).Flow plots of thrombomodulin (TM) and Tissue Factor (TF) expression onHUVECs cultured for 24 hours w/wo 10 ng/mL TNFα (G). Quantification ofthe change in TM and TF expression on HUVECs in response to 24 hourculture with increasing doses of TNFα (H). Flow plots of CD46 and CD59expression on HUVECs cultured for 24 hours w/wo 10 ng/mL TNFα (I).Quantification of CD46, CD59, and CD55 expression on HUVECs in responseto 24 hour culture with increasing doses of TNFα (J, K, and L,respectively). Quantification of the change in TM and TF expression onHUVECs in response to 48 hour culture with increasing doses of ATRA (M).Quantification of CD46, CD59, and CD55 expression on HUVECs in responseto 48 hour culture with increasing doses of ATRA (N, O, and P,respectively). (not significant (n.s.), * p<0.05, **p<0.01, ***p<0.001).

FIGS. 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, and 10I. Primary T cellstimulation, gut homing phenotype induction by ATRA, and CD55 mediatedcostimulation of T cell activation. Time course of TNFα secretion uponstimulation of Naïve human CD4⁺ T cells in serum free media for P1, P2,and P4-P6 (A). Time course of IL-10 secretion upon stimulation of NaïveCD4⁺ T cells in serum free media for P1, P2, and P4-P6 (B). Time courseof IFNγ secretion upon stimulation of Naïve CD4⁺ T cells in serum freemedia for P1, P2, and P4-P6 (C). Surface expression of the gut-homingintegrin α4β7 in human T cells grown in serum free conditions w/wosupplementation with 10 nM ATRA (D). Surface expression of thegut-homing chemokine receptor CCR9 in Naïve human CD4⁺ T cells grown inserum free conditions w/wo supplementation with 10 nM ATRA (E). CD69expression after stimulation of control and patient T cells with theindicated dose of anti-CD3 antibody w/wo costimulation with anti-CD55(F). Percent of Control and patient T cells that upregulated CD25 andCD69 expression after stimulation anti-CD3 antibody w/wo costimulationwith anti-CD28 or anti-CD55 (G). CFSE dilution in control or patient Tcells stimulation with 10 μg/mL anti-CD3 and the indicatedco-stimulatory molecules (H). IL10 secretion of control and patientcells in response to varying stimuli (I).

FIGS. 11A, 11B, and 11C. (A) C3d deposition on patient and control Tcells were incubated in the presence or absence of 5% nHS with orwithout the addition of 100 μM Compstatin to block complementactivation. (B) Ratio of C3a to C4a in culture supernatants after 24hours of incubation with 5% nHS w/wo 100 μM compstatin. (C) Ratio of C5ato C4a in culture supernatants after 24 hours of incubation with 5% nHSor without 100 μM compstatin. ***=p<0.001.

FIGS. 12A and 12B. Model of CHAPLE syndrome molecular pathogenesisleading to PLE. In healthy individuals CD55 prevents complementactivation and generation of anaphylatoxins. T cells produce aninflammatory cytokine mileu that is balanced towards production of IL10and control of intestinal immune responses. CD55 further protects fromcomplement activation on other host cells, including prevention ofcomplement activation on endothelial cells. CD55 deficiency associatedwith CHAPLE syndrome results in increased complement activation andanaphylatoxin production. The lack of CD55 and signaling byanaphylatoxins produces an inflammatory environment characterized by lowIL-10 production and increased TNFα production. Lower IL-10 productionlikely allows for further immunological activation, while TNFα drives aninflammatory environment and endothelial cell changes characterized bythe down regulation of complement regulatory proteins CD46 and CD59 andskewing of tissue factor and thrombomodulin expression towards aprocoagulative state. Lack of CD55 on endothelial cells furtherexasperates the changes caused by TNFα production leading touncontrolled activation of the coagulation and complement cascades onendothelial surfaces. This directly leads to endothelial barrier damageand protein/fluid loss into interstitial spaces. The presence of highconcentrations of retinoic acid in intestinal tissues drives theupregulation of CD55 and down regulation of thrombomodulin, likelyaccounting for the predisposition to intestinal manifestations in CHAPLEsyndrome (A). Proinflammatory and procoagulant changes described in A,and various pathological processes driven by excessive complementeffectors generated by immune response to the gut-microbiome lead tolymph vessel distortion and compromised lymphatic flow. Ensuinglymphangiectasia results in GI protein loss and malabsorption of fat andmicronutrients. Aggravation of the mucosal edema due to hypoproteinemiaand the pathological inflammation drives worsening enteropathy.Intraluminal complement activation likely impacts intestinal epithelialfunction. This, combined with hypercoagulability within blood vesselsimpairs circulatory dynamics thus aggravating the PLE (B).

DETAILED DESCRIPTION

As described herein, individuals with a CD55 gene mutation are afflictedwith CD55 deficiency, hyperactivation of complement, angiopathicthrombosis and protein losing enteropathy (CHAPLE), which includesdebilitating symptoms such as inflammatory bowel disease, protein losingenteropathy (which can be associated with hypoalbuminemia),hypogammaglobulinemia, intestinal lymphangiectasia, and/or thromboticevents.

The present disclosure is related, in part, to the surprising andunexpected discovery individuals afflicted with the above symptoms havea CD55 deficiency due to at least one CD55 gene mutations that resultsin the near to complete lack of expression of CD55 protein and/or theexpression of a CD55 protein that has substantially diminishedfunctional activity or that is devoid of functional activity.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entireties. In the case of conflict,the present specification, including definitions, will control. Inaddition, the examples are illustrative only and not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of amino acids in which case each amino acid numberfalling within the range is provided), between the upper and lower limitof that range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

The following terms are used to describe the present invention. Ininstances where a term is not specifically defined herein, that term isgiven an art-recognized meaning by those of ordinary skill applying thatterm in context to its use in describing the present invention.

The articles “a” and “an” as used herein and in the appended claims areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article unless the context clearlyindicates otherwise. By way of example, “an element” means one elementor more than one element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.”

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from anyone or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, in certain methods described hereinthat include more than one step or act, the order of the steps or actsof the method is not necessarily limited to the order in which the stepsor acts of the method are recited unless the context indicatesotherwise.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not preclude use in a humanor animal.

The terms “co-administration” and “co-administering” or “combinationtherapy” refer to both concurrent administration (administration of twoor more therapeutic agents at the same time) and time variedadministration (administration of one or more therapeutic agents at atime different from that of the administration of an additionaltherapeutic agent or agents), as long as the therapeutic agents arepresent in the patient to some extent, preferably at effective amounts,at the same time. In certain preferred aspects, one or more of thepresent compounds described herein, are co-administered in combinationwith at least one additional bioactive agent, especially including ananticancer agent. In particularly preferred aspects, theco-administration of compounds results in synergistic activity and/ortherapy, including anticancer activity.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein and includestautomers, regioisomers, geometric isomers, and where applicable,stereoisomers, including optical isomers (enantiomers) and otherstereoisomers (diastereomers) thereof, as well as pharmaceuticallyacceptable salts and derivatives (including prodrug forms) thereof whereapplicable, in context. Within its use in context, the term compoundgenerally refers to a single compound, but also may include othercompounds such as stereoisomers, regioisomers and/or optical isomers(including racemic mixtures) as well as specific enantiomers orenantiomerically enriched mixtures of disclosed compounds. The term alsorefers, in context to prodrug forms of compounds which have beenmodified to facilitate the administration and delivery of compounds to asite of activity. It is noted that in describing the present compounds,numerous substituents and variables associated with same, among others,are described. It is understood by those of ordinary skill thatmolecules which are described herein are stable compounds as generallydescribed hereunder. When the bond is shown, both a double bond andsingle bond are represented within the context of the compound shown.

The term “polypeptide” encompasses two or more naturally occurring orsynthetic amino acids linked by a covalent bond (e.g., an amide bond).Polypeptides as described herein include full length proteins (e.g.,fully processed proteins such as antibodies) as well as shorter aminoacids sequences (e.g., fragments of naturally occurring proteins orsynthetic polypeptide fragments such as antigen-binding fragments ofantibodies).

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human or a domesticated animal, to whomtreatment, including prophylactic treatment, with the compositionsaccording to the present disclosure is provided. For treatment of thoseconditions or disease states which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal. In general, in the present disclosure, the term patient refersto a human patient unless otherwise stated or implied from the contextof the use of the term.

The term “effective” is used to describe an amount of a compound,composition or component which, when used within the context of itsintended use, effects an intended result. The term effective subsumesall other effective amount or effective concentration terms, which areotherwise described or used in the present application.

The term “inflammatory bowel disease” refers to The term“lymphangiectasia” refers to a pathologic dilation of lymph nodes, whichcauses a disease known as “intestinal lymphagiectasia.”

The term “intestinal lymphangiectasia” refers to a disease characterizedby lymphatic vessel dilation, chronic diarrhea, and loss of proteins,such as serum albumin and globulin. Considered to be the cause of achronic form of protein-losing enteropathy.

The term “enteropathy” refers to any pathology of the intestine.

The term “protein losing enteropathy” (PLE) refers to any condition ofthe gastrointestinal tract (e.g., damage to the gut wall) that resultsin a net loss of protein from the body. Symptoms include, e.g.,diarrhea, fever, general abdominal discomfort, hypoproteinemia, and/oredema. See, also, the discussion of PLE above.

The term “thrombosis” refers to the formation of a blood clot inside ablood vessel, thereby obstructing blood flow through the circulatorysystem.

The term “thrombotic events” refers to the formation of a blood clotinside a blood vessel, thereby obstructing blood flow through thecirculatory system

The term “thromboembolism” refers to the obstruction of a blood vesselby a blood clot that has become dislodged from another site in thecirculatory system.

The term “hypoproteinemia” refers to a condition in which there is anabnormally low level of protein in the blood and is often accompaniedwith edema.

The term “angiopathic thrombosis” (or microangiopathic hemolytic anemia)refers to the pathology that relates from thrombosis in small bloodvessels (such as capillaries and arterioles). The thrombosis is usuallya consequence of endothelial cell injury, which results in fibrinexudation, fibrinoid necrosis, platelet aggregation, and thus, thethrombosis of small blood vessels

The term “hypogammaglobulinemia” refers to a type of hypoproteinemia, inwhich the level of gamma globulins is abnormally low, which results inprimary immune deficiency disease.

The term “hypoalbuminemia” refers to a type of hypoproteinemia in whichthe level of albumin in the blood is abnormally low.

The term “hyperactivation of complement” refers to a medical sign inwhich the complement pathway is overly active.

The term “disease” refers to any condition that impairs the normalfunctioning of the body. As such, diseases are associated withdysfunctioning of the body's normal homeostatic processes.

The term “syndrome” refers to the association of several medical signs,symptoms, and/or other characteristics that often occur together, whichmay have a single or a multifactorial cause.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multi-specific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

“Antibody fragments” (i.e., an antigen-binding fragment of an antibody),as defined for the purpose of the present disclosure, comprise a portionof an intact antibody, generally including the antigen binding orvariable region of the intact antibody and optionally the Fc region ofan antibody. Examples of antibody fragments include linear antibodies,single-chain antibody molecules (e.g., scFv), F(ab′)₂ fragments, Fab′fragments, and multi-specific antibodies formed from antibody fragments.The antibody fragments may retain at least part of the hinge andoptionally the C_(H)1 region of an IgG heavy chain. The antibodyfragments may retain the entire constant region of an IgG heavy chain,and include an IgG light chain.

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. Although the boundaries of the Fc region ofan immunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheFc region of an immunoglobulin generally comprises two constant domains,C_(H)2 and C_(H)3.

The term “F(ab) fragment” is defined as a fragment of an immunoglobulinmolecule that comprises the variable regions of a light chain and aheavy chain. That is, a Fab fragment is a monovalent antigen bindingstructure of an immunoglobulin without the Fc portion, and which resultsfrom the treatment of an immunoglobulin with papain.

The term “F(ab)′ fragment” is defined as a fragment of an immunoglobulinmolecule that comprises the variable regions of a light chain and aheavy chain. That is, the fragment is monovalent and most of the Fcportion is removed, which can be achieved through the treatment of animmunoglobulin molecule with pepsin and β-mercaptoethanol.

The term “F(ab′)₂ fragment” is defined as a fragment of animmunoglobulin molecule that comprises two F(ab) fragments and a portionof the hinge region. That is, most of the Fc portion is removed, whichcan be achieved through the treatment of an immunoglobulin molecule withpepsin.

The term “single-chain variable fragments” (scFvs) is defined as apolypeptide engineered to comprise the variable regions (i.e., theantigen-binding domains) of a light immunoglobulin chain and a heavyimmunoglobulin chain. The light chain and heavy chain can be joined by aflexible linker sequence.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present disclosure may bemade by the hybridoma method first described by Kohler and Milstein 1975Nature 256:495, or may be made by recombinant DNA methods. The“monoclonal antibodies” may also be isolated from phage antibodylibraries.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain and/or those residues from a “hypervariable loop” (i.e., residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptides will be prepared by at least one purification step.

In an aspect, the disclosure provides a method of diagnosing a patientor subject with CD55 deficiency, hyperactivation of complement,angiopathic thrombosis and protein losing enteropathy (CHAPLE). Themethod comprises: providing a sample from a patient; performing an assayto detect at least one of: at least one mutation in a DNA sequence of aCD55 gene, at least one mutation in a RNA sequence of a CD55 transcriptas compared to wild-type, at least one mutation in a DNA sequence of aCD55 complementary DNA (cDNA), decay-accelerating factor (DAF) or CD55protein, complement deposition, or a combination thereof; and diagnosingthe patient with CHAPLE. In an embodiment, the mutations in the DNAsequence of the CD55 gene or cDNA or the mutation in the RNA sequence ofthe CD55 RNA/transcript results in diminished CD55 protein expression(e.g., substantially diminished or devoice of CD55 expression) ordiminished or non-functional CD55 protein function (e.g., a CD55 thathas diminished or that is incapable of binding C3b and/or C4b).

In an embodiment, the patient is diagnosed with CHAPLE when at least oneof the following is detected: (i) at least one mutation in a DNAsequence, an RNA sequence or a cDNA sequence of CD55 that results in aCD55 protein with substantially diminished functional activity, a CD55protein with no functional activity, a lack of expression of CD55protein (i.e., no CD55 protein expression), or a substantiallydiminished expression of CD55 protein; (ii) a CD55 protein withsubstantially diminished functional activity; (iii) a CD55 protein withno functional activity; (iv) a lack of expression of CD55 protein (i.e.,no CD55 protein expression); (v) a substantially diminished expressionof CD55 protein; or (vi) a combination thereof.

In another embodiment, the patient is diagnosed with CHAPLE when thepatient has at least one CHAPLE related symptom and at least one of thefollowing is detected: (i) at least one mutation in a DNA sequence, anRNA sequence or a cDNA sequence of CD55 that results in a CD55 proteinwith substantially diminished functional activity, a CD55 protein withno functional activity, a lack of expression of CD55 protein (i.e., noCD55 protein expression), or a substantially diminished expression ofCD55 protein; (ii) a CD55 protein with substantially diminishedfunctional activity; (iii) a CD55 protein with no functional activity;(iv) a lack of expression of CD55 protein (i.e., no CD55 proteinexpression); (v) a substantially diminished expression of CD55 protein;or (vi) a combination thereof.

The sample can be any sample that would contain genome DNA (gDNA) orthat normally contains CD55 (i.e., a sample in which CD55 isfound/expressed in an individual without CHAPLE). For example, thesample may be a blood sample, such as peripheral blood mononuclear cells(PBMC). Providing a sample can comprise at least one of: obtaining asample from a patient (e.g., at least one PBMC), isolating gDNA from apatient sample, isolating (and/or purifying) protein from a patientsample or a combination thereof.

The CHAPLE associated symptom can include at least one of the followingsymptoms: inflammatory bowel disease, enteropathy, protein losingenteropathy, protein losing enteropathy associated with hypoalbuminemia,hypoalbuminemia, hypogammaglobulinemia, intestinal lymphangiectasia,lymphangiectasia, thrombotic events, thromboembolism, hyperactivation ofcomplement, angiopathic thrombosis, hypoproteinemia, or a combinationthereof. For example, the patient may have at least one, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, at least eleven, atleast twelve or all thirteen of the symptoms. In particular embodiments,the patient may have at least one of the following symptoms:inflammatory bowel disease, protein losing enteropathy associated withhypoalbuminemia, hypogammaglobulinemia, intestinal lymphangiectasia,thrombotic events or a combination thereof. For example, the patient canhave at least two of the symptoms, at least three of the symptoms, atleast four of the symptoms, or all five of the symptoms. That is, incertain embodiments, the patient has inflammatory bowel disease, proteinlosing enteropathy associated with hypoalbuminemia,hypogammaglobulinemia, intestinal lymphangiectasia, and thromboticevents.

The method can further comprise administering an effective amount of acomposition of the present disclosure to the subject with CHAPLE,wherein the composition is effective in treating or preventing at leastone symptom of CHAPLE. For example, the composition may comprise atleast one complement inhibitor. The complement inhibitor can be selectedfrom the group consisting of at least one serine protease inhibitor, atleast one soluble complement regulator, at least one therapeuticantibody or antigen-binding fragment thereof, at least one complementcomponent inhibitor, and at least one anaphylatoxin receptor antagonist.

In a further embodiment, the method further includes administering atleast one additional agent selected from the group consisting of ananticoagulant or a thrombolytic agent, somatostatin analogues (e.g.,octreotide), glucocorticoids, mesalizine, another immunosuppressiveagent (e.g., TNF-α blocker), an albumin transfusion, intravenousimmunoglobulins or a combination thereof. The additional agent can beincluded within the composition where appropriate or co-administered, asdiscussed above.

In an embodiment, the method of diagnosing a patient with CHAPLE canfurther comprise confirming the diagnosis by administering thecomposition of the present disclosure, thereby treating at least onesymptom of CHAPLE (i.e., a CHAPLE related symptom) or preventing thereoccurrence of at least one symptom of CHAPLE.

The mutation of the present disclosure, i.e., mutations in the DNAsequence of the CD55 gene (which can be determined or detected by amutation in the RNA sequence of the CD55 transcript (e.g., mRNA) or theDNA sequence of CD55 cDNA), can result in a near to complete absence ofCD55 protein expression or the expression of CD55 protein withsubstantially diminished function or that is non-functional. That is,the mutant CD55 protein can have diminished (or decreased) functionrelative to wild-type CD55. For example, mutant CD55 may bind C4b and/orC3b with lesser (or decreased) affinity and/or avidity, or not at all.In certain embodiments, CD55 is not present or substantially not presentin patients with CHAPLE syndrome/disease.

As discussed above, the mutation in the DNA sequence of the CD55 gene(and therefore, the RNA sequence of the transcript and the DNA sequenceof the cDNA of the CD55 gene) can be any mutation that results in: (i)the expression of CD55 protein with decreased function relative towild-type CD55 (e.g., CD55 protein with substantially no ability to bindC3b and/or C4b); and/or (ii) a decreased amount of CD55 expressionrelative to wild-type CD55 (e.g., no expression of CD 55 protein). Forexample, the mutation in the DNA sequence of the CD55 gene (which oneskilled in the art would appreciate that these mutations can be detectedin the RNA and DNA sequences of CD55 transcripts and cDNA, respectively)can be at least one of c.149-150delAA, c.149-150insCCTT, c.109delC,c.800G>C, c.287-1G>C, c.149-150delAAinsCCTT or a combination thereof.That is, in an embodiment, the DNA sequence of the CD55 gene has atleast one, at least two, at least three, or all four of theabove-mentioned mutations in the DNA sequence of the CD55 gene, or therelated transcript or cDNA.

In further embodiments, detecting can include at least one of: (i)sequencing at least a portion of the CD55 gene or the CD55 transcript orthe CD55 cDNA (or amplicons produced from the CD55 gene, transcript, orcDNA); or (ii) contacting a labeled nucleic acid probe (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more probes) to at least a portion of the CD55gene or the CD55 transcript or the CD55 cDNA (or amplicons produced fromthe CD55 gene, transcript, or cDNA); or (iii) contacting at least aportion of the CD55 gene or CD55 transcripts or the CD55 cDNA (oramplicons produced from the CD55 gene, transcript, or cDNA) with amicroarray; or (iv) a combination thereof.

Hybridization of the labeled nucleic acid probe brought into contactwith CD55 gDNA, RNA transcripts, or cDNA (or amplicons produced from theCD55 gene, transcript, or cDNA) from the patient's sample can beindicative of a mutation in the portion of the CD55 gene or CD55transcript or the CD55 cDNA. Alternatively, hybridization of the labelednucleic acid probe that was brought into contact with CD55 gDNA, RNAtranscripts, or cDNA (or amplicons produced from the CD55 gene,transcript, or cDNA) from the patient's sample can be indicative of theportion of the CD55 gene or CD55 transcript or the CD55 cDNA having awild-type sequence at (or lacking CD55 associated mutations in theportion hybridized) the location of hybridization.

Sequencing at least a portion of the CD55 gene (or the CD55 transcriptor CD55 cDNA that is produced from the CD55 transcripts) can includeamplifying at least one region of interest for sequencing. The region ofinterest for sequencing can include any coding sequence (exon) of theCD55 gene or any CD55 exon and portions that flank the exons. Forexample, in a particular embodiment, at least one polymerase chainreaction (PCR) is performed with at least one of the following primersets to produce amplicons of the CD55 gene, mRNA (e.g., transcripts),and/or cDNA: (i) CTACTCCACCCGTCTTGTTTGT and TTTGGGGGTTAAGGATACAGTC (Exon1); (ii) CAGGTGTGGCATTTCAAGG and ACCCTGGGGTTTAGTAACGC (Exon 2); (iii)AAGTACTAAATATGCGCAAAGCAG and ATGGTCCTATCAAGAAACATCC (Exon 3); (iv)GTTACCTTCTTTGTGTGTATGCC and GCTGTGAATACCAGTCATGAAAC (Exon 4); (v)AACCTGGAGAATTTGAGGAAAG and TGTGCTAATATTCTTAAGGGGC (Exon 5); (vi)GCATTTATAAGCATCTCTTGTTGG and TCATTGAATGTCTGCAACCC (Exon 6); (vii)CTAGGTGTTTGTGGGGAGAGAG and TCTGGTGGGTTTCTGAAGAGTT (Exon 7); (viii)TTTACGCAGAGTCCTTCAGC and CCATTTAATCCTGCAATCTTGG (Exon 8); (ix)TGGAAATTTGAGTTGCTTTCG and TCTCCCAGGAATATGGATTG (Exon 9); (x)GCACCCCAAATTAACTGATTC and ATGTGATTCCAGGACTGCC (Exon 10); or (xi) acombination thereof.

Contacting a labeled nucleic acid probe to at least a portion of theCD55 gene (or the CD55 transcript or the CD55 cDNA produced from theCD55 transcript, or amplicons produced from the CD55 gene, transcript,or cDNA) can be performed or accomplished with real-time PCR.Hybridization of a real-time PCR probe can be indicative of a mutationin the portion of the CD55 gene or CD55 transcript or CD55 cDNA (oramplicons produced from the CD55 gene, transcript, or cDNA).Alternatively, hybridization of a real-time PCR probe can be indicativeof the portion of the CD55 gene or CD55 transcript or CD55 cDNA (oramplicons produced from the CD55 gene, transcript, or cDNA) having awild-type sequence at the location of hybridization (or ampliconsproduced from the CD55 gene, transcript, or cDNA). Furthermore,contacting a labeled nucleic acid probe to at least a portion of theCD55 cDNA (or amplicons produced from the CD55 transcripts or cDNA) maycomprise: isolating CD55 transcripts; reverse transcribing at least aportion of the CD55 transcripts; and contacting the cDNA with thelabeled nucleic acid probe. This may further comprise amplifying aportion or region of interest via PCR (see discussion above) prior tocontacting the amplicons produced from CD55 cDNA with the labelednucleic acid probe.

The microarray can include probes (e.g., immobilized probes) designed orconfigured to detect or bind CD55 gene (i.e., the sense and/or antisenseDNA stand(s)), transcripts (i.e., mRNA), or cDNA sequence (or ampliconsproduced from the CD55 gene, transcript, or cDNA) of a patient with atleast one mutation that result in the complete absence in CD55 proteinor a CD55 protein with substantially diminished or devoid of function,as described above. Alternatively, the microarray can include probes(e.g., immobilized probes) designed or configured to detect or bind awild-type CD55 DNA, transcript (i.e., RNA), or cDNA sequence (oramplicons produced from the CD55 gene, transcript, or cDNA) of a patientor subject. In a further embodiment, the microarray may include probesdesigned or configured to individually detect (i.e., separately detect)both CD55 mutations and wild-type CD55.

As used herein, the terms “probe(s)” and “primer(s)” refers tooligonucleotide sequences that may readily be prepared based on the CD55nucleic acids described herein. A probe may comprise an isolated nucleicacid attached to a detectable label or reporter molecule. In any aspector embodiment described herein, the detectable label or label isselected from the group consisting of an enzyme, a chemiluminescentagent, a ligand, biotin, streptavidin, a radioactive molecule, and animmunofluorescent protein or dye. Methods for labeling and guidance inthe choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al. (1989) and Ausubel et al. (1987). “Primers” areshort nucleic acids, such as DNA oligonucleotides 15 nucleotides or morein length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides in length). Primers may be annealed toa complementary target DNA strand by nucleic acid hybridization to forma hybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (1989), Ausubel et al. (1987), and Innis etal., (1990). PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). One of skill in the art will appreciate that thespecificity of a particular probe or primer increases with its length.Thus, for example, a primer comprising 20 consecutive nucleotides of thehuman CD55 cDNA, RNA, or gene will anneal to a-target sequence such as aCD55 gene within a genomic DNA sample with a higher specificity than acorresponding primer of only 15 nucleotides. Thus, in order to obtaingreater specificity, probes and primers may be selected that comprise20, 25, 30, 35, 40, 50 or more consecutive nucleotides that arecomplementary to the nucleotide sequence at the site of a mutation asdescribed herein or the wild-type sequence at the same location.

Thus, the probe or probes of the present disclosure can include anucleic acid sequence that is complementary (e.g., hybridizes understringent conditions) to the site of a mutation as described herein orthe wild-type nucleic acid sequence at the same location. The probe caninclude complementary bases 5′ and 3′ of the site of mutation. Forexample, the probe includes complementary bases 5′ of the site ofmutation by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46, 47, 48, 49, or50 nucleotides (e.g., the probe include complementary bases 5′ of thesite of mutation by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46, 47, 48, 49, or50 nucleotides). For example, the probe includes complementary bases 3′of the site of mutation by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46,47, 48, 49, or 50 nucleotides (e.g., the probe include complementarybases 3′ of the site of mutation by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 46,47, 48, 49, or 50 nucleotides). As described above, in certainembodiments, the probe is complementary to the wild-type sequence,wherein no hybridization is indicative of a mutation and/orhybridization is indicative of no mutation. In other embodiments, theprobe is complementary to a mutation as described herein, whereinhydridization is indicative of a mutation and/or no hybridization isindicative of no mutation. As described above, the probe can becomplementary to the mRNA, the DNA sense strand, the DNA antisensestrand, and/or cDNA.

In any aspect or embodiments described herein, the nucleic acid probesand/or primes of the disclosure hybridize specifically to targetpolynucleotides of interest (i.e., a wild-type nucleic acid sequence ora mutation containing nucleic acid sequence as described herein) understringent conditions. Two molecules hybridizing to each other understringent conditions is an indication that the two nucleic acidsequences are substantially identical. Stringent conditions are sequencedependent and different under different environmental parameters and fordifferent assays. As used herein, “stringent conditions” or “stringenthybridization conditions” means any conditions in which hybridizationwill occur when there is at least about 95%, at least 96%, at least 97%,at feat 98%, or at least 99% nucleotide complementarity or identity(e.g., about 95% to about 100% or about 97 to about 100%, such as about95%. about 96%. about 97%, about 98%, about 99%, or about 100%) betweenthe nucleic acids (e.g., a polynucleotide of interest and a nucleic acidprobe). For example, stringent conditions for may be selected to beabout 5° C. to 20° C. lower than the thermal melting point (T_(m)) thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) and Tijssen (1993) and are otherwise known inthe art.

For example, appropriate high stringent hybridization conditions formicroarray may include, e.g., hybridization in a buffer, such as6×SSPE-T (0.9 M NaCl, 60 mM NaH₂PO₄, 6 mM EDTA and 0.05% Triton X-100)for between about 10 minutes and about at least 3 hours (e.g., at leastabout 15 minutes) at a temperature ranging from about 4° C. to about 37°C. In an embodiment, hybridization under high stringent conditions iscarried out in 5×SSC, 50% dionized Formamide, 0.1% SDS at 42° C.overnight.

Hybridization specificity can be evaluated by comparing thehybridization of specificity-control nucleic acid probes tospecificity-control target polynucleotides that are added to a sample ina known amount. The specificity-control target polynucleotides may haveone or more sequence mismatches compared with the corresponding nucleicacid probes. In this manner, whether only complementary targetpolynucleotides are hybridizing to the nucleic acid probes or whethermismatched hybrid duplexes are forming is determined.

Hybridization reactions can be performed in absolute or differentialhybridization formats. In the absolute hybridization format, targetpolynucleotides from one sample are hybridized to the probes in an array(e.g., in a microarray format) and signals detected after hybridizationcomplex formation correlate to target polynucleotide levels in a sample.In the differential hybridization format, the differential expression ofa set of genes in two biological samples is analyzed. For differentialhybridization, target polynucleotides from both biological samples areprepared and labeled with different labeling moieties. A mixture of thetwo labeled target polynucleotides is added to an array (e.g., amicroarray). The array is then examined under conditions in which theemissions from the two different labels are individually detectable.Probes in the array that are hybridized to substantially equal numbersof target polynucleotides derived from both biological samples give adistinct combined fluorescence (Shalon et al. PCT publicationWO95/35505). In a preferred embodiment, the labels are fluorescentlabels with distinguishable emission spectra.

After hybridization, the array (e.g., microarray) is washed to removenonhybridized polynucleotides and complex formation between thehybridizable array elements and the target polynucleotides is detected.Methods for detecting complex formation are well known to those skilledin the art. In a preferred embodiment, the target polynucleotides arelabeled with a fluorescent label and levels and patterns of fluorescenceindicative of complex formation are measured. In one embodiment, themeasurement is accomplished by fluorescence microscopy, preferablyconfocal fluorescence microscopy. An argon ion laser excites thefluorescent label, emissions are directed to a photomultiplier and theamount of emitted light detected and quantitated. The detected signalshould be proportional to the amount of probe/target polynucleotidecomplex at each position of the microarray. The fluorescence microscopecan be associated with a computer-driven scanner device to generate aquantitative two-dimensional image of hybridization intensity. Thescanned image is examined to determine the abundance/expression level ofeach hybridized target polynucleotide. In another embodiment, themeasurement of levels and patterns of fluorescence is accomplished witha fluorescent imaging device, such as a microarray scanner (e.g., Axonscanner with GenePix Pro software). As with the previous measurementmethod, the measurements can be used to determine theabundance/expression level of each hybridized target polynucleotide,thereby determining whether there is a mutation as described hereinand/or the expression level of CD55 dependening upon the techniqueutilized.

Detecting CD55 protein can be performed via any known protein detectiontechnique, e.g., western blot, flow cytometry, an immunoassay,microscopy, protein immunostaining, immunoelectrophoresis,spectrophotometry, etc. For example, detecting CD55 protein cancomprise: contacting the sample with at least one CD55 bindingpolypeptide. The CD55-binding polypeptide can be an anti-CD55 antibodyor a CD55-binding fragment thereof. The CD55-binding polypeptide may beused in any assay that can detect either a wild-type CD55 protein or amutant CD55 protein that has substantially diminished functionalactivity, for example, the CD55 has decreased or no binding activity forC3b and/or C4b (discussed in greater detail above), or both. The CD55binding polypeptides can include a detectable label. For example, thelabelled CD55 binding polypeptide can include a fluorescent label or anenzyme, which facilitates the detection of the CD55 protein (mutantand/or wild-type, as discussed above) via flow cytometry orELISA/western blot, respectively. One skilled in the art wouldappreciate that the CD55-binding polypeptide may be utilized in numerousother techniques, e.g., see above and below, to detect mutant CD55protein, wild-type CD55 protein, or both mutant and wild-type CD55protein.

Detecting CD55 protein can further comprise contacting the sample or aCD55-CD55-binding polypeptide complex with at least one secondarypolypeptide that binds specifically to the CD55 binding polypeptide. Thesecondary polypeptide can include a detectable label. The secondarypolypeptide can be an antibody or fragment thereof that binds the CD55binding polypeptide. For example, the labelled secondary polypeptide caninclude a fluorescent label or an enzyme, which facilitates thedetection of the CD55 protein (mutant and/or wild-type, as discussedherein) via flow cytometry or ELISA/western blot, respectively.

The present disclosure contemplates the use of any suitable detectionassay, which is known or becomes known to those skilled in the art. Aswould be appreciated by the skilled artisan in view of the presentdisclosure, any particular detection assay could be employed with nomore than routine experimentation. In certain embodiments, the step ofdetecting CD55 protein is performed using at least one of a westernblot, flow cytometry, an immunoassay, or a combination thereof. Forexample, the immunoassay can be at least one assay selected from thegroup consisting of enzyme-linked immunosorbent assay (ELISA), flowcytometry, radioimmunoassay, magnetic immunoassay, enzyme-linkedimmunospot (ELISPOT), and immunofluorescence. For example, detectingCD55 protein can be performed using a western blot of protein isolatedfrom (e.g., a crude or purified protein isolate) the patient sample(e.g., PBMCs) or flow cytometry performed on, e.g., PBMCs, in which CD55is detected with a CD55-binding polypeptide, which includes a detectablelabel (or marker) or via at least one secondary polypeptide thatincludes a detectable label (or marker). Alternatively, a sandwich (orcapture) ELISA could be utilized, in which the PBMCs are captured by,e.g., an antibody known to bind the surface of a PBMC (or a CD55 bindingprotein) thereby capturing the PBMCs, and detecting CD55 protein throughthe binding of a CD55-binding polypeptide. The CD55-binding polypeptidecan include a detectable label or a secondary polypeptide with adetectable label may be used, as discussed herein.

In another embodiment, the detecting of CD55 binding function includesexamining at least one of C3b affinity, C3b avidity, C4b affinity, C4bavidity or a combination thereof. That is, CD55 binding to C3b and/orC4b may be assessed by determining the affinity and/or avidity of theCD55-C3b or CD55-C4b interaction/binding. Examination of this functioninteraction (e.g., affinity and/or avidity) can be performed by methodswell-known in the art with routine experimentation for one skilled inthe art.

In a particular embodiment, detecting complement deposition includesdetecting C3d deposition. For example, the patient sample (e.g., PBMCs)may be contacted with a C3d binding polypeptide. The Cd3 bound Cd3binding polypeptide can include a detectable label (or marker) or thecomplex may be detected with a secondary polypeptide that is specificfor the Cd3 binding polypeptides and has a detectable label. As such,Cd3 may be detected by any of the methods articulated for detectingCD55, such as flow cytometry or ELISA.

In a further aspect, the disclosure provides a method of treating apatient having CD55 deficiency, hyperactivation of complement,angiopathic thrombosis and protein losing enteropathy (CHAPLE) orpreventing at least one symptom of CHAPLE in a patient at risk ofdeveloping the same. The method comprises administering an effectiveamount of a composition comprising at least one (e.g., at least two, atleast three, at least four, or at least five) complement inhibitor(e.g., one, two, three, four, five, six, seven, eight, nine, or tencomplement inhibitors) to a subject in need thereof, wherein thecomposition is effective in treating and/or preventing at least onesymptom of CHAPLE.

In some embodiments, the complement inhibitor is at least one of aserine protease inhibitor, a soluble complement regulator, a therapeuticantibody or an antigen-binding fragment thereof, a complement componentinhibitor (e.g., a small molecule), an anaphylatoxin receptorantagonist, or a combination thereof.

In certain embodiments, the serine protease inhibitors is at least oneof a C3 convertase inhibitor, a C5 convertase inhibitor, a C1 inhibitor,a C1r inhibitor, a C1s inhibitor, a C2a inhibitor, a MASP-1 inhibitor, aMASP-2 inhibitor, a factor D inhibitor, a factor B inhibitor, a factor Iinhibitor or a combination thereof. For example, the serine proteaseinhibitor can be at least one of the metBCX-1470 (BioCryst, Birmingham,Ala., USA), C1s-INH-248 (Knoll/Abbott, Abbott Park, Ill., USA),compstatin, Cetor® (Sanquin, Amsterdam, Netherlands), Berinert® (CSLBehring, King of Prussia, Pa., USA), Cinryze™ (ViroPharma, Exton, Pa.,USA), rhC 1INH (Pharming Group N.V., Leiden, Netherlands), Ruconest®(Salix Pharmaceuticals, Inc., Raleigh, N.C., USA) or a combinationthereof. Eculizumab is a recombinant humanized monoclonal antibody toC5, which inhibits the cleavage of C5 to C5a and C5b by the C5convertase. As such, eculizumab is a terminal complement inhibitor thatprevents C5b-9 formation (see, e.g., U.S. Pat. No. 9,409,980).Eculizumab was approved by the US Food and Drug Administration (FDA) in2007 for the treatment of PNH and in 2011 for the treatment of aHUS.

In other embodiments, the soluble complement regulator is at least oneof a soluble form of a membrane cofactor protein (MCP or CD46), asoluble form of a decay-accelerating factor (DAF or CD55), a solubleform of a membrane attack complex-inhibitor protein (MAC-IP or CD59), asoluble form of complement receptor 1 (CD35) or a combination thereof.For example, the soluble complement regulator can be at least one ofsCR1 (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sCR1-sL^(ex) (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge, Mass., USA), amembrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or a combinationthereof.

In further embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one polypeptide that binds C3, C3a, C3b, C3convertase, C5, C5a, C5b, C5 convertase, C7, C8, or C9, factor B, factorD, C4, C2, C1, properdin, a functional blocking antibody of ananaphylatoxin or a combination thereof. The binding can inhibitcomplement activation by at least one of blocking association/bindingwith other complement proteins, blocking association/binding withreceptor proteins, blocking serine protease activity or a combinationthereof. For example, the therapeutic antibody or the antigen-bindingfragment thereof can be at least one of eculizumab (Soliris®; AlexionPharmaceuticals Inc., New Haven, Conn., USA), ALXN1007 (AlexionPharmaceuticals Inc., New Haven, Conn., USA), neutrazumab (G2 Therapies,Darlinghurst, NSW, Australia), Pexelizumab (Alexion PharmaceuticalsInc., New Haven, Conn., USA), ofatumumab (Genmab A/S, Copenhagen,Denmark), HuMax-CD38 (Benmab A/S, Copenhagen, Denmark), TNX-558 (Tanox,South San Francisco, Calif., USA), TNX-234 (Tanox, South San Francisco,Calif., USA), TA106 (Taligen, Aurora, Colo., USA), anti-properdin(Novelmed, Cleveland, Ohio, USA) or a combination thereof. Thetherapeutic antibody or antigen-binding fragment thereof, may be alinear antibodies, single-chain antibody molecules (e.g., scFv), F(ab′)₂fragments, Fab′ fragments, or multi-specific antibodies formed fromantibody fragments. Furthermore, the antibody or antigen-bindingfragment thereof may be a humanized antibody, e.g. a humanized anti-C5antibody. Additionally, the antibody may be a monoclonal antibody, suchas an anti-C5 monoclonal antibody. For example, the antibody may be amonoclonal, humanized anti-C5 antibody.

In yet other embodiments, the complement component inhibitor is apeptide, nucleic acids, a synthetic molecule or a combination thereofthat disrupts protein functions by steric hindrance or the induction ofconformational changes. For example, the complement component inhibitorcan be at least one of compstatin, anti-C5 RNA aptamer (ARC1905;Archemix, Cambridge, Mass., USA), or analogs or derivatives thereof, ora combination thereof.

In another embodiment, the anaphylatoxin receptor antagonist is at leastone of a C5aR antagonist, a C5L2 antagonist, a C3a receptor antagonist,a functional blocking antibody of an anaphylatoxin or a combinationthereof. For example, the anaphylatoxin receptor antagonist is at leastone of PMX-53 (PepTech Corp, Bedform, Mass., USA), PMX-205 (PepTechCorp, Bedform, Mass., USA), JPE-1375 (Jerini, Berlin, Germany), JSM-7717(Jerini, Berlin, Germany), rhMBL (Enzon Pharmaceuticals, Cranford, N.J.,USA) or a combination thereof.

In another aspect, the disclosure provides a composition for treating orpreventing at least one symptom of CD55 deficiency, hyperactivation ofcomplement, angiopathic thrombosis and protein losing enteropathy(CHAPLE). The composition comprises an effective amount of two or moreagents, wherein at least one of the agents is a complement inhibitor toa subject in need thereof, and the composition is effective in treatingor preventing at least one symptom of CHAPLE. In an embodiment, theleast two of the agents are a complement inhibitor, e.g., a C3convertase inhibitor and a C5 convertase inhibitor. In anotherembodiment, the effective amount is a synergistically effective amount.In another embodiment, the composition is a therapeutic compositionfurther comprising a pharmaceutically acceptable carrier.

In an additional embodiment, the complement inhibitor is selected fromthe group consisting of a serine protease inhibitor, a solublecomplement regulator, a therapeutic antibody or an antigen-bindingfragment thereof, a complement component inhibitor, and an anaphylatoxinreceptor antagonist, each as described herein.

In further embodiments, the complement inhibitors include a C3convertase inhibitor and a C5 convertase inhibitor. In other embodimentsthe complement inhibitors include a soluble form of a CD55 and at leastone of a C3 convertase inhibitor, a C5 convertase inhibitor or acombination thereof.

In certain embodiments, the serine protease inhibitors is at least oneof a C3 convertase inhibitor, a C5 convertase inhibitor, a C1 inhibitor,a C1r inhibitor, a C1s inhibitor, a C2a inhibitor, a MASP-1 inhibitor, aMASP-2 inhibitor, a factor D inhibitor, a factor B inhibitor, a factor Iinhibitor or a combination thereof.

In a particular embodiment, the serine protease inhibitor is at leastone of BCX-1470 (BioCryst, Birmingham, Ala., USA), C1s-INH-248(Knoll/Abbott, Abbott Park, Ill., USA), compstatin, Cetor® (Sanquin,Amsterdam, Netherlands), Berinert® (CSL Behring, King of Prussia, Pa.,USA), Cinryze™ (ViroPharma, Exton, Pa., USA), rhC1INH (Pharming GroupN.V., Leiden, Netherlands), Ruconest® (Salix Pharmaceuticals, Inc.,Raleigh, N.C., USA) or a combination thereof.

In other embodiments, the soluble complement regulator is at least oneof a soluble form of a membrane cofactor protein (MCP or CD46), asoluble form of a decay-accelerating factor (DAF or CD55), a solubleform of a membrane attack complex-inhibitor protein (MAC-IP or CD59), asoluble form of complement receptor 1 (CD35) or a combination thereof.

In an embodiment, the soluble complement regulator is at least one ofsCR1 (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sCR1-sL^(ex) (TP10; Advant Immunotherapeutics, Needham, Mass., USA),sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge, Mass., USA), amembrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or a combinationthereof.

In other embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one polypeptide that binds C3, C3a, C3b, C3convertase, C5, C5a, C5b, C5 convertase, C7, C8, or C9, factor B, factorD, C4, C2, C1, properdin, a functional blocking antibody of ananaphylatoxin or a combination thereof, wherein said binding inhibitscomplement activation by at least one of blocking association/bindingwith other complement proteins, blocking association/binding withreceptor proteins, blocking serine protease activity or a combinationthereof.

In certain embodiments, the therapeutic antibody or the antigen-bindingfragment thereof is at least one of eculizumab (Soliris®; AlexionPharmaceuticals Inc., New Haven, Conn., USA), ALXN1007 (AlexionPharmaceuticals Inc., New Haven, Conn., USA), neutrazumab (G2 Therapies,Darlinghurst, NSW, Australia), Pexelizumab (Alexion PharmaceuticalsInc., New Haven, Conn., USA), ofatumumab (Genmab A/S, Copenhagen,Denmark), HuMax-CD38 (Benmab A/S, Copenhagen, Denmark), TNX-558 (Tanox,South San Francisco, Calif., USA), TNX-234 (Tanox, South San Francisco,Calif., USA), TA106 (Taligen, Aurora, Colo., USA), anti-properdin(Novelmed, Cleveland, Ohio, USA) or a combination thereof.

In yet other embodiments, the complement component inhibitor is apeptide, nucleic acids, a synthetic molecule or a combination thereofthat disrupts protein functions by steric hindrance or the induction ofconformational changes.

In a particular embodiment, the complement component inhibitor is atleast one of compstatin, anti-C5 RNA aptamer (ARC1905; Archemix,Cambridge, Mass., USA), or analogs or derivatives thereof, or acombination thereof.

In additional embodiments, the anaphylatoxin receptor antagonist is atleast one of a C5aR antagonist, a C5L2 antagonist, a C3a receptorantagonist, a functional blocking antibody of an anaphylatoxin or acombination thereof.

In certain embodiments, the anaphylatoxin receptor antagonist is atleast one of PMX-53 (PepTech Corp, Bedform, Mass., USA), PMX-205(PepTech Corp, Bedform, Mass., USA), JPE-1375 (Jerini, Berlin, Germany),JSM-7717 (Jerini, Berlin, Germany), rhMBL (Enzon Pharmaceuticals,Cranford, N.J., USA) or a combination thereof.

Therapeutic Compositions

Pharmaceutical compositions comprising combinations of an effectiveamount of at least two agents (e.g., therapeutic agents), wherein one ormore of the agents is a complement inhibitor, all in effective amounts(e.g., synergistically effective amounts), in combination with apharmaceutically effective amount of a carrier, additive or excipient,represents a further aspect of the present disclosure.

The present disclosure includes, where applicable, the compositionscomprising the pharmaceutically acceptable salts, in particular, acid orbase addition salts of the compounds (e.g., agents and/or complementinhibitors) of the composition described herein. The acids which areused to prepare the pharmaceutically acceptable acid addition salts ofthe aforementioned base compounds useful according to this aspect arethose which form non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions, such as the hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, acetate, lactate, citrate, acid citrate, tartrate,bitartrate, succinate, maleate, fumarate, gluconate, saccharate,benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate and pamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3naphthoate)]salts, among numerous others.

Pharmaceutically acceptable base addition salts may also be used toproduce pharmaceutically acceptable salt forms of the compounds orderivatives according to the present disclosure. The chemical bases thatmay be used as reagents to prepare pharmaceutically acceptable basesalts of the present compounds that are acidic in nature are those thatform non-toxic base salts with such compounds. Such non-toxic base saltsinclude, but are not limited to those derived from suchpharmacologically acceptable cations such as alkali metal cations (e.g.,potassium and sodium) and alkaline earth metal cations (e.g., calcium,zinc and magnesium), ammonium or water-soluble amine addition salts suchas N-methylglucamine-(meglumine), and the lower alkanolammonium andother base salts of pharmaceutically acceptable organic amines, amongothers.

The compounds as described herein may, in accordance with thedisclosure, be administered in single or divided doses by the oral,parenteral or topical routes. Administration of the active compound mayrange from continuous (intravenous drip) to several oral administrationsper day (for example, Q.I.D.) and may include oral, topical, parenteral,intramuscular, intravenous, sub-cutaneous, transdermal (which mayinclude a penetration enhancement agent), buccal, sublingual andsuppository administration, among other routes of administration.Enteric coated oral tablets may also be used to enhance bioavailabilityof the compounds from an oral route of administration. The mosteffective dosage form will depend upon the pharmacokinetics of theparticular agent chosen as well as the severity of disease in thepatient. Administration of compounds according to the present disclosureas sprays, mists, or aerosols for intra-nasal, intra-tracheal orpulmonary administration may also be used. The present disclosuretherefore also is directed to pharmaceutical compositions comprising aneffective amount of compound as described herein, optionally incombination with a pharmaceutically acceptable carrier, additive orexcipient. Compounds according to the present disclosure may beadministered in immediate release, intermediate release or sustained orcontrolled release forms. Sustained or controlled release forms arepreferably administered orally, but also in suppository and transdermalor other topical forms. Intramuscular injections in liposomal form mayalso be used to control or sustain the release of compound at aninjection site.

The compositions as described herein may be formulated in a conventionalmanner using one or more pharmaceutically acceptable carriers and mayalso be administered in controlled-release formulations.Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as prolaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions as described herein may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions as described herein may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such as Ph. Helv orsimilar alcohol.

The pharmaceutical compositions as described herein may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions as described herein maybe administered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient, which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions as described herein may also beadministered topically. Suitable topical formulations are readilyprepared for each of these areas or organs. Topical application for thelower intestinal tract can be effected in a rectal suppositoryformulation (see above) or in a suitable enema formulation.Topically-acceptable transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this disclosure include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. In certain preferred aspects of the disclosure, the compounds maybe coated onto a stent which is to be surgically implanted into apatient in order to inhibit or reduce the likelihood of occlusionoccurring in the stent in the patient.

Alternatively, the pharmaceutical compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions as described herein may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of compound in a pharmaceutical composition as describedherein that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host and diseasetreated, the particular mode of administration. Preferably, thecompositions should be formulated to contain between about 0.05milligram to about 750 milligrams or more, more preferably about 1milligram to about 600 milligrams, and even more preferably about 10milligrams to about 500 milligrams of active ingredient, alone or incombination with at least one other compound according to the presentdisclosure.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease or condition beingtreated.

A patient or subject in need of therapy using compounds according to themethods described herein can be treated by administering to the patient(subject) an effective amount of the compound according to the presentdisclosure including pharmaceutically acceptable salts, solvates orpolymorphs, thereof optionally in a pharmaceutically acceptable carrieror diluent, either alone, or in combination with other agents asotherwise identified herein.

These compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, including transdermally, in liquid, cream,gel, or solid form, or by aerosol form.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated. A preferred doseof the active compound for all of the herein-mentioned conditions is inthe range from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kgper day, more generally 0.5 to about 25 mg per kilogram body weight ofthe recipient/patient per day. A typical topical dosage will range from0.01-5% wt/wt in a suitable carrier.

The compound is conveniently administered in any suitable unit dosageform, including but not limited to one containing less than 1 mg, 1 mgto 3000 mg, preferably 5 to 500 mg of active ingredient per unit dosageform. An oral dosage of about 25-250 mg is often convenient.

The active ingredient is preferably administered to achieve peak plasmaconcentrations of the active compound of about 0.00001-30 mM, preferablyabout 0.1-30 μM. This may be achieved, for example, by the intravenousinjection of a solution or formulation of the active ingredient,optionally in saline, or an aqueous medium or administered as a bolus ofthe active ingredient. Oral administration is also appropriate togenerate effective plasma concentrations of active agent.

The concentration of the active compound in the drug composition willdepend on absorption, distribution, inactivation, and excretion rates ofthe drug as well as other factors known to those of skill in the art. Itis to be noted that dosage values will also vary with the severity ofthe condition to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

Oral compositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it can contain, in addition to material of the abovetype, a liquid carrier such as a fatty oil. In addition, dosage unitforms can contain various other materials which modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents.

The active compound or pharmaceutically acceptable salt thereof can beadministered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active compound or pharmaceutically acceptable salts thereof canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action. In certainpreferred aspects of the disclosure, one or more compounds according tothe present disclosure are coadministered with another bioactive agentincluding an antibiotic, as otherwise described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811 (which isincorporated herein by reference in its entirety). For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound is then introduced into the container. The container is thenswirled by hand to free lipid material from the sides of the containerand to disperse lipid aggregates, thereby forming the liposomalsuspension.

The present disclosure also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, preferably a human. By “systemicadministration” is meant in vivo systemic absorption or accumulation ofdrugs in the blood stream followed by distribution throughout the entirebody. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged polymer is desired to bedelivered to). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms which prevent thecomposition or formulation from exerting its effect.

Administration routes which lead to systemic absorption include, withoutlimitations: intravenous, subcutaneous, intraperitoneal, inhalation,oral, intrapulmonary and intramuscular. The rate of entry of a drug intothe circulation has been shown to be a function of molecular weight orsize. The use of a liposome or other drug carrier comprising thecompounds of the present disclosure can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation which canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful.

The present disclosure also features the use of the compositioncomprising surface-modified liposomes containing poly (ethylene glycol)lipids (PEG-modified, or long-circulating liposomes or stealthliposomes). Polypeptides and compositions of the present disclosure canalso comprise covalently attached PEG molecules of various molecularweights. These formulations offer a method for increasing theaccumulation of drugs in target tissues. This class of drug carriersresists opsonization and elimination by the mononuclear phagocyticsystem (MPS or RES), thereby enabling longer blood circulation times andenhanced tissue exposure for the encapsulated drug (Lasic et al. Chem.Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43,1005-1011). Long-circulating liposomes are also likely to protect drugsfrom nuclease degradation to a greater extent compared to cationicliposomes, based on their ability to avoid accumulation in metabolicallyaggressive MPS tissues such as the liver and spleen. All of thesereferences are incorporated by reference herein.

The present disclosure also includes compositions prepared for storageor administration which include a pharmaceutically effective amount ofthe desired compounds (e.g., agents and/or complement inhibitors) in apharmaceutically acceptable carrier or diluent. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated byreference herein. For example, preservatives, stabilizers, dyes andflavoring agents can be provided. These include sodium benzoate, sorbicacid and esters of p-hydroxybenzoic acid. In addition, antioxidants andsuspending agents can be used.

An effective amount, pharmaceutically effective dose, therapeuticallyeffective amount, or pharmaceutically effective amount is that doserequired to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) of a diseasestate or pathological condition. The effective amount depends on thetype of disease, the composition used, the route of administration, thetype of mammal being treated, the physical characteristics of thespecific mammal under consideration, concurrent medication, and otherfactors which those skilled in the medical arts will recognize.Generally, an amount between 0.1 mg/kg and 1000 mg/kg body weight/day ofactive ingredients is administered dependent upon potency of thenegatively charged polymer. In addition, effective amounts of thecompositions of the disclosure encompass those amounts utilized in theexamples to facilitate the intended or desired biological effect.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds (e.g., agents and/or complement inhibitors) that exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects. The data obtained from the cell culture assays andanimal studies can be used in formulating a range of dosage for use inhumans. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the present disclosure, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The formulations can be administered orally, topically, parenterally, byinhalation or spray or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising at least twoagents, wherein at least one is a complement inhibitor, and apharmaceutically acceptable carrier. For example, two or more complementinhibitors can be present in association with one or more non-toxicpharmaceutically acceptable carriers and/or diluents and/or adjuvants,and if desired other active ingredients. The pharmaceutical compositionsof the present disclosure can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed. Formulations fororal use can also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present. Pharmaceutical compositions of the presentdisclosure can also be in the form of oil-in-water emulsions. The oilyphase can be a vegetable oil or a mineral oil or mixtures of these.Suitable emulsifying agents can be naturally-occurring gums, for examplegum acacia or gum tragacanth, naturally-occurring phosphatides, forexample soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol, anhydrides, for example sorbitan monooleate,and condensation products of the said partial esters with ethyleneoxide, for example polyoxyethylene sorbitan monooleate. The emulsionscan also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.The composition can also be administered to a subject in combinationwith other therapeutic compounds to increase the overall therapeuticeffect. The use of multiple compounds to treat an indication canincrease the beneficial effects while reducing the presence of sideeffects.

A further object of the present disclosure is to provide a kitcomprising a suitable container, the therapeutic of the presentdisclosure in a pharmaceutically acceptable form disposed therein, andinstructions for its use.

Preparations for administration of the therapeutic of the presentdisclosure include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's intravenous vehicles including fluid and nutrient replenishers,electrolyte replenishers, and the like. Preservatives and otheradditives may be added such as, for example, antimicrobial agents,anti-oxidants, chelating agents and inert gases and the like.

The compounds or peptides (e.g., agent, agents, and/or complementinhibitors; also referred to herein as “active compounds”) of thedisclosure, and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the compound orprotein and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the present disclosure is formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e.,topical), transmucosal, intraperitoneal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufferssuch as acetates, citrates or phosphates, and agents for the adjustmentof tonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor™.(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups, or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound. For buccal administration the compositions may takethe form of tablets or lozenges formulated in conventional manner. Foradministration by inhalation, the compounds for use according to thepresent disclosure are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. The compounds mayalso be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides. In addition to the formulationsdescribed previously, the compounds may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Additional objects and advantages of the present disclosure will beappreciated by one of ordinary skill in the art in light of the currentdescription and examples of the preferred embodiments, and are expresslyincluded within the scope of the present disclosure.

Examples

Materials and Methods.

Study Participants.

Three initial patients, their unaffected parents, and then sevenadditional patients presenting with a similar clinical presentation wereevaluated (FIGS. 1A and 3). The patients were followed in MarmaraUniversity, Istanbul, Turkey; Cerraphasa University, Istanbul, Turkey;Baskent University, Ankara, Turkey; Gazi University, Ankara, Turkey, andSami Ulus Hospital, Ankara, Turkey. All of the patients were enrolled ina clinical protocol which had been approved by the institutional reviewboards of the respective institutions and NIH. All study participants ortheir parents provided written informed consent.

Genetic and Functional Analysis.

Whole exome DNA sequencing (WES) was performed on the initial threepatients and their unaffected parents and siblings followed by specificCD55 DNA sequencing in the other patients and family members. Complementdeposition assays, cytokine secretion and T-cell stimulation assays wereperformed on patient samples and healthy controls. Complement depositionwas verified using lentiviral and clustered regularly-interspaced shortpalindromic repeats (CRISPR) mediated CD55 knockdown/knockout in JurkatT cells and HT29 epithelial cells.

Human Subjects.

All human subjects (or their legal guardians) in this study providedwritten informed consent in accordance with Helsinki principles forenrollment in research protocols that were approved by the InstitutionalReview Boards of the National Institute of Allergy and InfectiousDiseases, National Institutes of Health (NIH) or the CeMM ResearchCenter for Molecular Medicine of the Austrian Academy of Sciences.Patient and healthy control blood was obtained at the respective Turkishinstitutions overseeing patient care under approved protocols, andshipped to either the NIH or the CeMM Research Center for MolecularMedicine of the Austrian Academy of Sciences. Additional healthy controlblood was obtained at the NIH clinical center under approved protocols.Mutations will be archived by Online Mendelian inheritance in Man(OMIM), and whole-exome data will be submitted in dbGaP. A webpagethrough the National Center for Biotechnology Information (NCBI) willalso be created to accumulate patient mutation data in the format of theLeiden Online Variant Database (LOVD) as patients are identified.

Genetic Analysis Methods.

Genomic DNA (gDNA) was obtained from probands and family members byisolation and purification from peripheral blood mononuclear cells(PBMCs) using DNeasy® Blood and Tissue Kit (QIAGEN®, Hilden, Germany).DNA was then submitted for Whole Exome Sequencing (WES) or targetingsequencing of the CD55 gene. For whole exome sequencing, the Human AllExon 50 Mb kit (Agilent Technologies, Santa Clara, Calif., USA) coupledwith massively parallel sequencing by Illumina® HiSeg™ Sequencing System(IIlumina, Inc., San Diego, Calif., USA) was performed using thecollected DNA. For individual samples, WES produced approximately50-100× sequence coverage for targeted regions. WES was performed onpatients 1.1, 2.1 along with 3.1 and 5.1 in which WES was combined withhomozygosity mapping. As described previously, all sequenced DNA readswere mapped to the hg19 human genome reference by Burrows-WheelerAligner with default parameters. Single nucleotide variant and indelcalling were performed using the Genome Analysis Toolkit (BroadInstitute, Cambridge, Mass., USA). All SNVs/indels were annotated bySeattleSeq Annotation and an in-house custom analysis pipeline was usedto filter and prioritize for autosomal recessive or de novodisease-causal variants based on the clinical pedigree for Patients 1.1and 2.1, the mutations were identified by targeted gene screening of theWES data based on the similarity of clinical phenotype in the cohort(Lucas C L, et al. Heterozygous splice mutation in PIK3R1 causes humanimmunodeficiency with lymphoproliferation due to dominant activation ofPI3K. J Exp Med 2014; 211: 2537-47).

For targeted sequencing, CD55/DAF exons 1 through 10 were PCR amplified,purified, or gel extracted using QIAGEN's MinElute® PCR Purification Kit(QIAGEN®, Hilden, Germany) or QIAquick® Gel Extraction Kit (QIAGEN®,Hilden, Germany), respectively. Samples were submitted to the NationalInstitute of Allergy and Infectious Disease Research Technologies BranchCore Sequencing facility for Sanger sequencing. DNA sequences wereanalyzed using SEQUENCHER® V.5.3 (Gene Codes Corporation, Ann Arbor,Mich., USA). The primers used for Sanger sequencing and the ampliconsproduced therefrom are shown in Table 1, below.

TABLE 1 Primer Sequences for mutation detection in CD55 SEQ SEQ Exon #Forward Primer Sequence ID N Reverse Primer sequence ID NO Exon 1CTACTCCACCCGTCTTGTTTGT  4 TTTGGGGGTTAAGGATACAGTC  5 Exon 2CAGGTGTGGCATTTCAAGG  6 ACCCTGGGGTTTAGTAACGC  7 Exon 3AAGTACTAAATATGCGCAAAGCAG  8 ATGGTCCTATCAAGAAACATCC  9 Exon 4GTTACCTTCTTTGTGTGTATGCC 10 GCTGTGAATACCAGTCATGAAAC 11 Exon 5AACCTGGAGAATTTGAGGAAAG 12 TGTGCTAATATTCTTAAGGGGC 13 Exon 6GCATTTATAAGCATCTCTTGTTGG 14 TCATTGAATGTCTGCAACCC 15 Exon 7CTAGGTGTTTGTGGGGAGAGAG 16 TCTGGTGGGTTTCTGAAGAGTT 17 Exon 8TTTACGCAGAGTCCTTCAGC 18 CCATTTAATCCTGCAATCTTGG 19 Exon 9TGGAAATTTGAGTTGCTTTCG 20 TCTCCCAGGAATATGGATTG 21 Exon 10GCACCCCAAATTAACTGATTC 22 ATGTGATTCCAGGACTGCC 23

Primary Cells and Cell Lines.

Patient or control blood was subjected to a ficoll density gradientcentrifugation, after which peripheral blood mononuclear cells (PBMCs)were collected from the interface. Naïve or total CD4⁺ T cells were thenisolated by negative selection using the Naive CD4⁺ T Cell Isolation KitII or the human CD4⁺ T cell isolation kit, respectively (Miltenyi BiotecInc., Auburn, Calif., USA). Jurkat E6, HEK 293T, HT29 and Caco-2 cellswere purchased from the American Type Culture Collection (ATCC®;Manassas, Va., USA). Pooled Human Umbilical Vein Endothelial Cells(HUVECs) were from Lonza® (Walkersville, Md., USA).

Media.

Human T cells and Jurkat T cells were either grown in either RPMI 1640(Gibco™/ThermoFisher Scientific, Waltham, Mass., USA) supplemented with10% heat-shocked FBS (Gibco™/ThermoFisher Scientific, Waltham, Mass.,USA), 1% Penicillin/Streptomycin, 1% L-Glutamine and 100units/mL IL-2(complete RPMI) or X-Vivo 15 (Lonza) supplemented with 1%Penicillin/Streptomycin, 1% L-Glutamine and 100units/mL IL-2 (completeX-vivo 15). HT29 and HEK293T cells were grown in DMEM media(Gibco™/ThermoFisher Scientific, Waltham, Mass., USA) supplemented with10% heat-shocked FBS (Gibco™/Thermo Fisher Scientific, Waltham, Mass.,USA), 1% Penicillin/Streptomycin, and 1% L-Glutamine. HUVECS werecultured in Endothelial Cell Basal Medium (Lonza®, Walkersville, Md.,USA) supplemented with the contents of EGM™ SingleQuot™ Kit Suppl. &Growth Factors kit (Lonza, Walkersville, Md., USA). Caco-2 cells werecultured in Minimum Essential Media (Gibco™, Waltham, Mass., USA)supplemented with 20% non-heat inactivated FBS (Sigma), 1%Penicillin/Streptomycin, 1% L-Glutamine, and 10 mM HEPES.

Antibodies and Inhibitors.

Anti-CD25 FITC, Anti-CD69 APC, Anti-CD4 PE, Anti-CD55 FITC, anti-CD59FITC, anti-CD46 APC, anti-CD141/Thrombomodulin APC, anti-CD142/ThrombinPE, anti-CCR9 APC, anti-integrin α4 PE, anti-integrin β7, anti-C5aRFITC, anti-C5L2 PE, and anti-C3aR APC were purchased from BioLegend®(San Diego, Calif., USA) or eBisociences, Inc. (San Diego, Calif., USA).The anti-C3d antibody used in complement deposition experiments waspurchased from Hycult® Biotech (Plymouth Meeting, Pa., USA). LIVE/DEAD™Fixable Near-IR Dead Cell Stain Kit used for cell viability assay waspurchased from ThermoFisher Scientific (Waltham, Mass., USA) and APClabeled Annexin-V was from BioLegend® (San Diego, Calif., USA). Theanti-CD3 antibody HIT3a used in T cell re-stimulation experiments waspurchased from BioLegend® (San Diego, Calif., USA). The anti-CD55antibody (clone BRIC 216) used to costimulate T cells was from EMDMillipore (Billerica, Mass., USA) or International Blood Group ReferenceLaboratory (IBGRL) (Filton, UK). Human CD97-fc recombinant protein usedfor T cell costimulation was purchased from Creative BioMart® (Shirley,N.Y., USA). The C3aR inhibitor, SB 290157, was purchased fromCalbiochem® (San Diego, Calif., USA). The C5aR1 inhibitor, NDT 9513727,was purchased from Tocris Bioscience (Bristol, UK). Anti-CD55 used forwestern blotting was from Sigma-Aldrich® (St. Louis, Mo., USA).

Flow Cytometry. Adherent cells were treated with 0.05% trypsin EDTA tolift them from the surface followed by addition of 5% FBS to inactivatethe trypsin. Suspension cells were collected from culture followingmixture of the culture by repeat pipetting. Cells were washed andresuspended in FACS buffer (1% FBS, 0.05% sodium azide, and 5 mM EDTA inPBS) at 2×10⁶ cells/mL. Staining or isotype antibodies were added at1:200 final dilutions and incubated with cells at 4° C. for 30 minutesto 1 hour. Cells were then washed 3× and resuspended in FACS buffer with1% PFA and analyzed by flow cytometry. Flow files were analyzed onFlowJo version 9.9 or above (FlowJo, LLC, Ashland, Oreg., USA).

Complement Deposition.

T cells (Patient CD4⁺ T cell blasts or Jurkat T cells) were collectedand washed 3× in basal RPMI. Cells were then resuspended in basal mediaand rested for 2-3 hours at 37° C. to allow any deposited complementcomponents to be cleared from the cell surface. Rested cells were thencollected and resuspended in basal RPMI or basal RPMI that had beenadjusted to pH 6.5 (acidified media) to a final concentration of 1×10⁶cells/mL. Non-heat shocked pooled normal human serum was then added tocell suspension for the indicated amount of time. Human serum that hadbeen heat shocked at 56° C. for 30 minutes was used as a negativecontrol for complement activation. At the indicated times cells weretransitioned to 4° C., and kept chilled for the remainder of theexperiment. Cells were stained with a 1:300 dilution of anti-C3dantibody for 30 minutes, washed 3× and then stained with an anti-ratantibody conjugated with alexa fluor 488 at a 1:500 dilution for 30minutes. Cells were washed 3× and resuspended in FACS buffer (PBS, 1%FBS) and analyzed for C3d deposition by flow cytometry. To analyze C3ddeposition on HT29 cells, cells were either left untreated or treatedovernight with 10 ng/mL TNFα to upregulate CD55 expression. Cells werethen treated with trypsin/EDTA to release cells into suspension, washed,and then analyzed for C3d deposition as above.

Preparation of T cell Stimulatory Surfaces.

Tissue culture treated 48, or 96 well plates were incubated with theindicated concentration of anti-CD3 in PBS for 2 hours at 37° C. Plateswere then washed 3× in PBS and incubated with either anti-CD28,anti-CD55, or CD97fc for 2 hours at 37° C. Plates were again washed 3×with PBS before using in T cell stimulations.

T cell Stimulation and Cytokine Secretion.

To measure T cell activation isolated CD4 T cells were either left aloneor stained with 1 μM CFSE for 5 minutes at room temperature, protectedfrom light. Labelled cells were washed 3× in complete RPMI prior to useand resuspended at 1×10⁶ cells/mL in complete RPMI. Cells were thenadded to stimulatory surfaces, pre-coated with the indicatedconcentrations of activating antibodies/proteins. To measure CD25 andCD69 upregulation, non-CFSE labeled T cells were collected after 24hours and analyzed by flow cytometry for CD25 and CD69 expression, asdetailed above. To assess T cell proliferation, CFSE labeled T cellcultures were collected after 96 hours of stimulation and analyzed byflow cytometry for CFSE dilution.

For cytokine secretion upon primary stimulation, Naïve CD4⁺ T cells wereisolated via negative selection using the Naive CD4⁺ T Cell IsolationKit II, human isolation kit (Miltenyi Biotec Inc., Auburn, Calif., USA).Naïve T cells were then resuspended in complete X-Vivo 15 media.Dynabeads® CD3/CD28 T cell activator (Invitrogen™, Carlsbad, Calif.,USA) were then added to cells (1 bead/2 cells) and cells and dynabeadswere centrifuged at 300×g for 5 minutes to complex beads and T cells.Cells were resuspended and added to 48 well plates at a finalconcentration of 5×10⁴/mL in complete X-vivo 15 media. Cell culturesupernatants were collected every 24 hours starting at 96 hourspost-activation for the indicated time course. Cell cultures were storedat ⁻80° C. until analyzed. To analyze cytokine secretion uponrestimulation cells were collected from day 12 to 14 of culture incomplete X-Vivo 15 media, washed and resuspended in basal media at 37°C. for 3 hours to rest. Cells were then collected and resuspended incomplete X-Vivo 15 media and stimulated for 48 hours at 37° C. with 1μg/mL plate-bound anti-CD3. Cell supernatants were then collected andstored at ⁻80° C. until analyzed.

Lentiviral Construction and Transfection Protocol.

Lentiviral packaging constructs psPAX2 and PDM2.G were kind gifts of Dr.Nir Hacohen, Broad Institute. shRNA constructs against CD55 or emptyvector were purchased from Sigma-Aldrich® (St. Louis, Mo., USA). Togenerate lentiviral particles, 1.2×10⁶ HEK293T cells were seeded in eachwell of a six well plate the day prior to transfection. Cells were thencotransfected using lipofectamine 2000 (Invitrogen™, Carlsbad, Calif.,USA) with 900 ng of psPAX2 and 100 ng of pDM2.G, together with 1 μg ofthe vector of interest (shRNA or lentiCRISPRv2). Alternatively,lentivirus was produced by the conventional calcium chloridetransfection method. Supernatants were harvested 24 and 48 hours aftertransfection and frozen at ⁻80° C. HT29 cells were transduced by spininfection with lentivirus. Lentivirus and 8 μg/ml Polybrene(Sigma-Aldrich®, St. Louis, Mo., USA) were added to the wells of a24-well culture plate and centrifuged at 2000 rpm and 37° C. for 2hours. Lentivirus-containing media was then replaced with 293T media,and the cultures were maintained as described above. On day 5 ofculture, puromycin (Sigma-Aldrich®, St. Louis, Mo., USA) was added to afinal concentration of 2 μg/ml to select for virally transduced cells.Selected cells were maintained in 2 μg/mL puromycin following initialselection.

CRISPR Materials/Methods.

CRISPRs targeting CD55 were designed using an online tool(crispr.mit.edu) as previously described and cloned into a lentiCRISPRv2vector (Ran F A et al. Genome Engineering using the CRISPR-Cas9 system.Nature Protocols 2013; 8: 2281-308; and Sanjana N E, et al. Improvedvectors and genome-wide libraries for CRISPR screening. Nat Methods2014; 11: 783-4). CRISPR lentivirus was produced according to the aboveprotocol. Transductions were performed as above, with minor changes. ForJurkats, selection with 1 μg/mL puromycin was performed for 3 days. ForCaco-2, selection with 5 μg/mL puromycin was performed for 6 days,changing the media every 2 days.

HUVEC Experiments.

Huvecs grown to 80% confluency, and before passage 8, were trypsonizedand replated in 24 well tissue culture plates at 200,000 cells/well inHuvec media. Twenty-four hours later the indicated concentrations ofTNFα, IL10, or all trans retinoic acid (ATRA) were added. Twenty-fourhours later in the cast of TNFα or IL10 treatment or 48 hours later inthe case of ATRA, cells were trypsonized and stained for the indicatedsurface markers following the described flow cytometry stainingprotocol. Cells were then immediately analyzed.

Western Blotting.

CD4⁺ T cells were washed in PBS and lysed in 1% Triton X-100, 50 mMTris-C1, pH 8, 150 mM NaCl, 2 mM EDTA, 10% glycerol, complete proteaseinhibitor cocktail (Roche Diagnostics, Indianapolis, Ind., USA), andphosphatase inhibitor cocktails (Sigma-Aldrich®, St. Louis, Mo., USA) onice for 20 minutes. The lysates were then clarified by centrifugation at15,000×g at 4° C. for 10 minutes. Protein concentration was determinedby BCA assay (Thermo Fisher Scientific®, Waltham, Mass., USA). Lysateswere then diluted with 2× SDS sample buffer (quality biologicals)supplemented with 5% BME to make reducing sample buffer. Approximately 5μg of total protein was separated by SDS-PAGE on 4-20% precast gels(Invitrogen™, Carlsbad, Calif., USA) and transferred to a nitrocellulosemembrane (Bio-Rad Laboratories, Philadelphia, Pa., USA). Membranes wereblocked with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.01%Tween-20 (TBST) for 30 minutes at room temperature before incubatingwith primary antibody overnight at 4° C. After three 5 minute washeswith TBST at room temperature with rocking, HRP-conjugated secondaryantibody was added for an additional hour at room temperature. Afterfive 5 minute washes, HRP substrate (Luminata Forte; EMD Millipore,Billerica, Mass., USA) was added to the membranes, which were thenexposed to autoradiography film and developed.

Statistical Analysis.

Statistics were computed using the analysis options in Prism (GraphpadSoftware, Inc., La Jolla, Calif., USA). Either the Mann-Whitney U testor a two-tailed unpaired T test with Welch's correction was used tocompare sample means. *=p<0.05, **=p<0.01, ***=p<0.001.

Patient Clinical Histories.

Patient 1.1, a 7 year-old girl born to consanguineous Turkish parents,presented with bloody diarrhea and vomiting at 4 weeks of age, andcontinued with episodes throughout childhood. At 6 months of age, shewas admitted to hospital with persistent diarrhea, pneumonia, andfacial/extremity edema, for which she received antibiotics and albuminreplacement therapy. Endoscopic examination revealed ulcers, exudateformation and nodularity in the colon. Although the patient'sgastrointestinal (GI) symptoms improved with treatment and shesubsequently gained weight, long-term remission could not be achieved.The patient was placed on infliximab therapy at 6 years of age to dealwith ongoing GI symptoms. Apart from GI symptoms she experiencedrecurrent respiratory infections at an unusually high frequency from 6months of age. Immunologic evaluation revealed pan-hypogammaglobulinemiaand she was treated with intravenous immunoglobulin (IVIG), leading to adecrease in the frequency of infections. To determine the specificantibody production we tested isohemagglutin levels, with normal Anti-Aand Anti-B titers. Despite WIG, trough levels of IgG remained below thedesired range, and tracked well with levels of serum albumin (FIG. 1B).Fecal excretion of alpha-1 antitrypsin was found to be elevated,implying the role of protein losing enteropathy in refractoryhypogammaglobulinemia. In addition to the biallelic loss of function inCD55, this patient was additionally positive for a homozygous LOFmutation in CD21 that resulted in loss of protein expression (data notshown).

Patient 2.1, a 22-year-old female born to consanguineous Turkishparents, presented with fever, productive coughing and hemoptysis at theage of 6 years when she was diagnosed with pneumonia. She continued tohave recurring respiratory symptoms with breathing difficulty, wheezing,chronic coughing, and multiple attacks of pneumonia leading to thedevelopment of bronchiectasis and finger clubbing. Following the onsetof respiratory illness, she developed GI complaints, with abdominal painand distention, diarrhea, and, eventually, cessation of growth. Clinicalpresentation included vomiting, diarrhea, periorbital and pretibialedema and she was found to have hypoalbuminemia and decreased serumlevels of Vitamin B12, folate, iron, and hypothyroxinemia. When she was11 years old her serum immunoglobulins levels were determined due tosuspected immunodeficiency. These tests revealed hypogammaglobulinemiaand she was started on IVIG replacement therapy. While on IVIG thefrequency of infections decreased substantially, leading to the recoveryof bronchiectasis. Despite IVIG, serum immunoglobulin G levels remainedwithin the lower range and correlated with the serum albumin levels overtime, suggesting the loss of immunoglobulins through the GI tract (FIG.1B). In order to assess the patient's capacity to produce specificantibodies, we evaluated isohemaglutinin titers and antibody response topneumococcal vaccine, with normal results. After several years of lossto follow up, she presented to the gastroenterology clinic withpersisting symptoms and received treatment for IBD. Despite treatment,she experienced symptoms of intestinal obstruction at the age of 22years and underwent surgery for the resection of the narrowed intestinalsegment (FIG. 1H). The resection material showed fissuring ulcers,cryptitis and crypt abscesses, villous blunting, diffuse epithelialregeneration, and dramatic lymphangiectasis.

Patient 3.1, a 16-year-old boy born to consanguineous Turkish parents,presented with edema due to hypoalbuminemia at 27 months of age. Serialcolonoscopies revealed progressive macroscopic hyperemic lesions withulcers, cryptitis, crypt abcess, and lymphoid cell infiltration that didnot respond to steroids and azathioprine treatment alone. Over time,addition of mesalazine to treat IBD and supplementation therapy withcalcium, vitamin B12 and folic acid led to clinical improvement. Ofnote, the patient did not present with classical VEO-IBD symptoms suchas bloody stools at the beginning of the disease. The patient presentedwith protein losing enteropathy with hypoalbuminemia and serial serumimmunoglobulin investigation revealed reduced levels of IgG. Lymphocytecounts were also reported as normal. This patient has not, as of yet,presented with recurrent/severe lower respiratory tract infectionsdespite hypogammaglobulinemia. In addition, no infectious trigger wasfound throughout GI episodes indicated by normal C-reactive protein(C-RP) levels, as well as a normal erythrocyte sedimentation rate (ESR).

Patient 4.1, a 3-year-old girl born to consanguineous parents, presentedwith puffy eyes, diarrhea and vomiting at 1 year of age. She continuedto have recurrent episodes of periorbital and pretibial edema, mostlytriggered by the bouts of bloody and mucous diarrhea. She was found tohave persistent hypoalbuminemia, for which she received intermittentalbumin infusions. Evaluation for other serum proteins revealedhypogammaglobulinemia, with low IgG, IgA and IgM levels. Atpresentation, anthropometric indices revealed wasting with weight withinthe 3^(rd) to 10^(th) percentile and height within the 50^(th)percentile for her age. The patient currently receives supplementationtherapy for the deficiency of vitamin B12. Despite low immunoglobulinlevels she has not experienced any significant or recurrent infectionsas of yet. Evaluation for infectious etiology during attacks of diarrheaand vomiting proved negative, along with normal levels of inflammatorymarkers such as C-reactive protein (CRP) and erythrocyte sedimentationrate (ESR). Evaluation for the differential diagnoses including foodallergy and cystic fibrosis or celiac disease was also negative.

Patient 4.2, a 17 year-old boy and brother of Patient 4.1 and 4.3, has ahistory of occasional and self-limiting presentations with facial andextremity edema, for which no medical intervention was required. Despitetesting positive for hypogammaglobulinemia, he denied any history ofinfections of unusual severity or frequency. The patient has notexperienced any significant GI symptoms thus far.

Patient 4.3, an 18-year-old boy and brother of patient 4.1 and 4.2, hasbeen mostly asymptomatic. Laboratory evaluation revealed low-normallevels of serum proteins and vitamin B12. The patient has notexperienced any significant GI symptoms thus far, and has not reportedany history of edema.

Patient 4.4 was an affected female cousin of patients 4.1-4.3 with anunknown genotype. Clinically, this patient presented with recurrentfacial and extremity edema, chronic bloody diarrhea, chronicmalnutrition, low serum protein levels, with hypoalbuminemia (albumin:1.9 g/dl) and hypogammaglobulinemia. Histopathology revealed thepresence of dramatic lymphangiectasis in the small intestine. Thepatient was treated with immunosuppressive treatment including steroidsand azathioprine due to suspected IBD. Interestingly, she had a historyof thrombosis and cerebral vascular disease with hypodense brain lesionin the lateral ventricule and received anticoagulation therapy with lowmolecular weight heparin. Angiographic studies demonstrated variousvascular pathologies at the intra-abdominal and cerebral vesselsincluding: (1) narrowed superior mesenteric vein with multiplecollateral veins, suggestive of chronic thrombosis, and (2) decreasedblood flow in internal carotid arteries, particularly in cavernous andintracranial segments and undetectable middle cerebral artery. Sheeventually developed an intestinal obstruction and underwent intestinalresection surgery to remove the narrow segments. Histopathologicalevaluation of the resection material revealed lymphangiectasia, a focusof ulceration, and abundant ectatic vascular elements with vessel wallthickening. At 33 years of age, this patient died of thrombosis, cardiacarrhythmia, and respiratory distress syndrome developing after surgicalresection to remove a severe intestinal obstruction. We were unable tosuccessfully obtain a genetic analysis of this patient due to not havingaccess to patient tissue.

Patient 5.1 is a 10-year-old boy and the sibling of Patient 5.2 born toconsanguineous Turkish parents. The patient was first admitted to ahospital at the age of one year with diarrhea and vomiting. He wasdiagnosed with intestinal lymphangiectasia with severe hypoproteinemia,with low albumin and immunoglobulin levels. The patient requiredfrequent albumin infusions and was put on a protein rich diet withmedium-chain triglyceride (MCT) supplements. At the age of 7 years old,Patient 5.1 underwent abdominal surgery for the resection oflymphangiectatic segments, which led to a temporary recovery of hissymptoms. He has had multiple recurrent thrombotic events in mesentericand hepatic veins, as well as in the vena cava inferior, atrial andventricular thrombosis. He received anticoagulation treatment withwarfarin following cardiac surgery for atrial thrombosis. He alsopresented with intracranial bleeding and underwent neurosurgicaloperations. Currently, the patient is prescribed octreotide irregularly.The patient is not reported to have an increased rate of respiratoryinfection despite low immunoglobulin levels. There was no evidence ofgastrointestinal infection, and sweat chloride test, celiac markers, andlipid profiling were normal. Colonic biopsy revealed edema andeosinophilic infiltrates, from which a diagnosis of eosinophilic colitiswas made. The patient is in poor condition and requires frequenthospital admissions.

Patient 5.2, is the 12-year-old affected sister of Patient 5.1. Thepatient presented with frequent abdominal pain, and a similarmanifestation of hypogammaglobulinemia and hypoalbuminemia as Patient5.1, which required frequent albumin transfusions. Due to episodes ofintestinal obstruction, she underwent resection of edematous small bowelsegments to remove lymphangiectatic segments, upon which she experiencedsome clinical remission. Of note, this patient did not present with anythrombotic events, infectious history, or bloody diarrhea.

Patient 5.3, an affected sister of patients 5.1 and 5.2 of unknowngenotype, was admitted at 15 months old with complains of eyelid andextremity edema and diarrhea. The patient presented withhypoalbuminemia, hypogammaglobulinemia, thrombocytosis and anemia.Endoscopy and histopathological examination of the duodenum revealedintestinal lymphangiectasis. The patient had severe malnutrition, withreduced levels of calcium, magnesium, and phosphorus and variousvitamins. The patient was treated with intravenous calcium, albumin,vitamin, and mineral supplements on top of medium chain triglyceridesupplements and octreotide. These treatments led to temporary clinicalimprovement. Small bowel resection was performed three times over aperiod of 2 years with only transient clinical improvement. Parenteralnutrition efforts were interrupted by Candida and S. Aureus infectionsalong with sepsis. She developed a thrombus in the vena cava superiorleading to vena cava superior syndrome and anti-thrombotic treatmentwith low molecular weight heparin and tissue plasminogen activator wasinitiated. The patient developed ascites, pleural effusion, andpulmonary infection and died when she was 4.5 years old.

Patient 6.1, a 15-year-old boy born to consanguineous Turkish parents,presented with facial and extremity edema at 1.5 years of age and wasfound to have hypoalbuminemia. He had frequent hospital admissions dueto vomiting, diarrhea and abdominal pain. Endoscopic examinationrevealed intestinal lymphangiectasia and he was put on steroidtreatment. Evaluation for common causes of secondary lymphangiectasiaincluding heart diseases and abdominal mass lesions was negative. Uponpersistence of his symptoms he was started on octreotide treatment andprescribed a diet low in fat and high in high quality protein along withMCT supplements. Along with chronic gastrointestinal symptoms hedeveloped deficiencies in the major micronutrients and vitamins such asiron, calcium, magnesium and vitamin D leading to growth retardation.Frequent exacerbations of facial and extremity edema and abdominalsymptoms in relation to severe intestinal wall edema (FIG. 1C) requiredrepeated albumin transfusions. In line with the low albumin levels,serum immunoglobulins were also decreased (Table 2, shown as FIG. 4).Apart from chronic gastrointestinal symptoms he experienced frequentrespiratory symptoms with chronic cough and finger clubbing. Computedtomography of the chest revealed fibrotic changes in the posterobasallung segments. When he was 14 years of age, he suffered severethrombotic events, with a thrombus originating from a stalk 2 cm distalto the inferior vena cava (IVC) extending into the right atrium, whichwas impairing the venous blood flow. He also had thrombi in thepulmonary arteries. He underwent thoracic surgery for thrombectomy andwas commenced on anticoagulation treatment with low molecular weightheparin and low dose aspirin. Despite prophylactic anticoagulationtreatment, during the follow up he experienced recurrence of thrombosiswith reformation of the clot in the right atrium. Screening for commoncongenital hypercoagulable states such as Factor V Leiden or prothrombinG20210A mutations that lead to over activity of coagulation factors, orarising from a deficiency of the natural anticoagulants Protein C, S andanti-thrombin III was negative. The patient was found heterozygous forthe thermolabile variant of the methylenetetrahydrofolate reductase(MTHFR) gene, C677T and no A1298C mutation was observed in this genealong with normal plasma homocystein level, suggesting no increased riskof thrombosis attributable to hyperhomocysteinemia. The patient was alsotested for anticardiolipin and anti-phospholipid antibodies and forparoxysmal nocturnal hemoglobinuria through Fluorescein-labeledproaerolysin (FLAER) flow cytometry assay, with negative results. Thepatient currently suffers a debilitating disease with frequent hospitaladmissions.

Patient 7.1, a 4-year-old girl born to consanguineous Syrian parents,presented with facial and extremity edema at 1 year of age, withcontinued relapses thereafter. She has experienced recurrentgastrointestinal symptoms, with chronic diarrhea and was found to havehypoalbuminemia. She was diagnosed with suspected intestinallymphangiectasia and commenced on octreotide treatment. She has growthretardation, with height for age below the 3^(rd) percentile. Due tomicronutrient deficiencies she received supplementation therapy withvitamin D, vitamin B 12, multivitamins, and was transfused witherythrocyte suspension due to anemia. She was prescribed a diet low infat and high in high quality proteins, MCT supplements, and furthersupplementation by enteral feeding and albumin transfusion as required.Measurement of serum immunoglobulins revealed low IgG, IgA and IgM(Table 2). Immunologic evaluation revealed normal isohemaglutinin titersand the proportion of the lymphocyte subsets was within normal limits.She has experienced recurrent lower respiratory infections and receivedparenteral antibiotics and was put on prophylactic antibiotic treatmentwith co-trimoxazole.

Clinical Phenotype.

Demographic data and the clinical presentations of the 10 patients inthe study are shown in Table 2 (FIG. 4), and patient descriptions weredescribed above. Patients 1.1-3.1 were born to unaffected consanguineousparents with a distinct PLE syndrome comprising severe hypoalbuminemia,hypogammaglobulinemia and chronic diarrhea. After gene discovery inthese initial cases, additional patients were found by evaluatingearly-onset PLE patients.

Overall, patients presented with facial and extremity edema accompaniedby chronic persistent bloody diarrhea with remissions and exacerbationsin disease severity (Table 3). Individual patients had additionaldisease manifestations, some with multiple thrombotic events andvascular alterations and others having a history of recurrentrespiratory infections.

All patients exhibited persistent hypoproteinemia, with reductions bothin albumin and gammaglobulins (FIG. 1B and FIG. 5A). The degree ofhypoproteinemia and the severity of symptoms varied among individualcases. Patients 4.2 and 4.3 had mild disease with occasional facial andextremity edema whereas others had profound hypoalbuminemia and a severeprogressive disease course (Table 2). Radiological exams, taken frompatients in the presence of acute GI symptoms, showed that the patientshad bowel wall edema/thickening (FIG. 1C, arrows) sometimes togetherwith partial intestinal obstruction, which responded favorably toalbumin replacement therapy. The hypoproteinemia was attributed to GIprotein loss because the nadir of albumin levels correlated with theflares of bloody and mucous diarrhea and increased excretion of α-1antitrypsin was detected in feces in Patient 1.1. Other causes ofhypoproteinemia, including hepatic disease, decreased nutritionalintake, or heavy proteinuria, were excluded in all cases. Endoscopicevaluation revealed relatively mild alterations in the mucosa given theseverity of protein loss, whereas histopathology of small intestinalbiopsies demonstrated dilatation of the lacteals and distension of thelymphatic vessels in Patients 2.1, 5.1, 5.2, 6.1 and 7.1 (Table 2, FIG.1D). Evaluation for common systemic conditions that could lead tosecondary lymphangiectasia, such as cancer, heart disease or obstructivelesions involving the lymphatic system, was negative, indicating adiagnosis of primary intestinal lymphangiectasia as the cause of PLE.Surgical removal of the localized lymphangiectatic segments in Patients5.1 and 5.2 resulted in substantial, but temporary, reversal of PLE inthe case of Patient 5.1, and symptom alleviation in the case of Patient5.2.

The patients eventually developed an intestinal malabsorption syndrome,with anemia and major micronutrients deficiencies including iron,calcium, magnesium, folate and vitamins D and B12, ultimately leading toretarded growth (Table 2 and FIGS. 5B and 5C). Supplementation therapywith vitamins and micronutrients along with dietary modification andalbumin and blood transfusions as required led to improved growth insome of the patients, whereas others had a more refractory course withprogressive malnutrition (FIG. 5).

Some of the patients had hypercoagulability with recurrent thrombosisand vascular alterations. Patient 6.1, who initially had relativelystable GI disease, developed severe thrombotic disease during follow up.Large occlusive thrombi occurred in the inferior vena cava (IVC) andright atrium together with thrombi in the pulmonary arteries (FIG. 1E,left panel), which was accompanied by arteriovenous malformations in thelung (FIG. 1E, center and right panels). Despite surgical removal ofblood clots and anticoagulation therapy, continuing thrombosis caused anoverall worsening of symptoms. Similarly, Patient 5.1 had multiplethrombotic events in the mesenteric and hepatic veins as well as theright atrium and ventricle, which was treated with low molecular weightheparin anticoagulation. He also had a history of intracranialhemorrhage, which required cranial surgery. Of note, a cousin ofPatients 4.1-4.3 and a sibling of Patients 5.1 and 5.2 (FIG. 3), withundetermined genotypes, had a similar course of disease leading to deathat 33 and 4.5 years of age respectively. These patients presented withPLE with lymphangiectasia leading to malnutrition, and a history ofthrombotic events, which in Patient 4.4 led to cerebral ischemia,mesenteric vein narrowing with development of multiple collateralvessels and histopathologic evidence of ectasia of the vascular elementsin the submucosa of the small intestine. Patient 4.4 died of cardiac,thrombotic, and respiratory complications following surgery to remove anintestinal obstruction, while Patient 5.3 died following development ofascites, pleural effusion, and pulmonary infection.

Some of the patients had endoscopic and histopathology featuresresembling Crohn's disease although without granulomas. Patients 1.1-3.1showed mucosal ulcers and inflammatory exudate in the terminal ileum andcolonic sites (FIG. 1F). In the endoscopic biopsy materials fromPatients 1.1, 2.1, and 3.1, there were mixed infiltrates of T and Bcells, sometimes with eosinophils (FIG. 1G). Although these threepatients received recommended therapy for IBD that reduced their bowelsymptoms, a sustained remission was not achieved and GI protein losscontinued (Table 2, FIG. 1B, and Figure. 5). Patient 2.1, who recentlydeveloped symptoms of intestinal obstruction, underwent surgicalresection of a narrowed ileal segment (FIG. 1H) which included dilatedlymphatic vessels, leading to resolution of GI symptoms, weight gain andrecovery of serum albumin levels during the short term follow up (notshown). None of the patients exhibited extraintestinal manifestationscommonly associated with IBD, such as skin eruptions, perianal lesions,fistulas or uveitis.

Patients 1.1, 2.1, 6.1, and 7.1 experienced recurrent respiratoryinfections in association with hypogammaglobulinemia (Table 2, FIG. 1B(arrows)), while other patients did not report any significant orrecurrent infections. As major immunological subsets and specificantibody production were largely normal in the patients (Table 3), it isbelieve these infections were largely due to loss of serumimmunoglobulins. Clinical evaluation of patient peripheral bloodimmunological subsets of Table 3, provided as either (cells/mm³) or % oftotal cells. Notably, use of intravenous immunoglobulin (IVIG)replacement therapy decreased the frequency of infections in Patients1.1 and 2.1 and led to resolution of bronchiectasis in Patient 2.1.

TABLE 3 Lymphocyte subset characterization in CHAPLE patient peripheralblood Patient ID P 1.1 P 2.1 P 3.1 P 4.1 P 4.2 P 4.3 P 5.1 P 5.2 P 6.1 P7.1 Lymphocyte Subsets (cells/mm³) or % of total cells CD3⁺ 7400 226278% 1552 1742 1748 56% 80% 75% 78% (900-4500) (800-3500) (55-78)(900-4500) (800-3500) (800-3500) (55-78) (55-78) (52-78) (43-76)CD3⁺CD4⁺ 3630 1222 28%  994  962  874 27% 44% 49% 37% (500-2400)(400-2100) (27-53) (500-2400) (400-2100) (400-2100) (27-53) (27-53)(25-48) (23-48) CD3⁺CD8⁺ 3080  832 39%  534  676  879 24% 21% 28% 37%(300-1600) (200-1200) (19-34) (300-1600) (200-l200) (200-1200) (19-34)(19-34) (9-35) (14-33) CD19⁺  94  156 24%  378  364  179 29%  8%  6% 14%(200-2100) (100-500) (10-31) (200-2100) (100-500) (100-500) (10-31)(10-31) (8-24) (14-44) CD3⁺  198  78 12%  120  390  176 11%  7%  1%  3%CD16⁺/CD56⁺ (100-1000) (90-600) (4-26) (100-1000) (90-600) (90-600)(4-26) (4-26) (6-27) (4-23)

Identification of CD55 Mutations.

Given the consanguinity in all families under study, an autosomalrecessive (AR) mode of inheritance was assumed. WES in Patients 1.1 and2.1, and combined with homozygosity mapping in Patient 3.1 and 5.1 asdescribed previously (Dobbs K, et al., Inherited DOCK2 Deficiency inPatients with Early-Onset Invasive Infections. The New England Journalof Medicine 2015; 372: 2409-22), revealed potentially deleterioushomozygous loss-of-function (LOF) mutations in CD55/Decay AcceleratingFactor (DAF), showing perfect segregation under assumption of ARinheritance. CD55 is a negative regulator of the complement cascadeubiquitously expressed on the surface of eukaryotic cells (FIG. 6A). TheCD55 nucleotide variants were confirmed by Sanger DNA sequencing (FIG.3). Additional patients were then screened with a diagnosis of PLE andidentified 5 additional patients from Families 4, 6, and 7 with rarebiallelic CD55 LOF nucleotide variants. Patient 1.1 and 7.1 werehomozygous for dinucleotide deletion and 4 nucleotide insertions atposition c.149-150 and patients 2.1, 3.1, 5.1 and 5.2 were homozygousfor a single base pair deletion at position c.109, each resulting inframeshift and premature termination codons in CD55. In Family 4, anovel homozygous missense mutation was identified (c.800G>C) in CD55resulting in a single amino acid substitution (p.Cys267Ser) in the4^(th) short consensus repeat (SCR) domain, predicted by computationalanalyses to be deleterious. In Family 6, a mutation was found in thesplice acceptor site of exon 3. The locations of the identifiedmutations within the CD55 protein/gene are depicted in FIG. 6B, anddeleterious predictions are shown in Table 4. Mutation and impact scoreof Table 4 were calculated from indicated predictive algorithm (eitherthe SIFT, Polyphen2 and CADD algorithms). This scoring system was notapplicable (N/A) to frameshift or splice site mutations present inmajority of our patients. Predictions were based on a reference sequencefrom ENST00000367064, GRCh37. Altogether, we identified 4 distincthomozygous, novel rare mutations in CD55 in a total of 11 patients ofTurkish (2 with unknown genotype), and 1 of Syrian origin (FIG. 6B).

TABLE 4 Predicted impact scored for identified CD55 varients MutationChromosome Position SIFT Polyphen2 CADD c.149_150delAAinsCCTT 1207495774 N/A N/A 5.585 c.109delC 1 207495734 N/A N/A 12.37 c.800G > C 1207504588 0 0.99 (probably 23.1 (deleterious) damaging) c. 287-1G > A 1207497903 N/A N/A 23.9

Effects of CD55 Mutations on Messenger RNA and Protein Expression.

Patients 1.1, 2.1, 3.1, 5.1, and 5.2 harbored frameshift mutations thatled to nonsense-mediated loss of mRNA in 1.1 and 2.1 (FIG. 6C). Themutation present in Patients 4.1, 4.2, and 4.3 is a missense mutation(p.Cys267Ser) that does not alter mRNA expression level (FIG. 6C).Cys267 participates in an intrachain disulfide bond with Cys225 and lossof this bond could lead to CD55 protein misfolding and degradation(FIGS. 7A and 7B) (Nakano Y, et al. Complete Determination of disulfidebonds localized within the short consensus repeat units of decayaccelerating factor (CD55 antigen). Biochim Biophys Acta 1992; 1116:235-40). Absent, or severely reduced, CD55 surface expression wasobserved by flow cytometry or western blot in all patients (FIGS. 6D and6E). Patient 6.1, harboring a splice acceptor site mutation, is the onlypatient with residual protein expression by flow, possibly suggestingexon skipping.

Increased Complement Deposition on CD55-Deficient Cells.

As CD55 is a negative regulator of the complement pathway, it washypothesized that CD55 deficiency would lead to increased C3 cleavageand deposition on host cells. Activation of the alternative pathway,marked by C3 cleavage, can be enhanced by serum acidification, usedpreviously for the diagnosis of paroxysmal nocturnal hemoglobinuria(PNH) (Ham T H, et al., Studies on Destruction of Red blood Cells. Li.Chronic Hemolytic Anemia with Paroxysmal Nocturnal Hemoglobinuria:Certain Immunological Aspects of the Hemolytic Mechanism with SpecialReference to Serum Complement. J. Clin Invest 1939: 18: 657-72). Hence,control CD4⁺ T-cell blasts, which highly express CD55 on their cellsurface (FIG. 6D), were compared with those from patients afterincubation with normal or acidified pooled normal human serum (nHS),both of which caused surface deposition of the complement split productC3d. We observed that C3d was deposited to a greater degree on T cellsfrom multiple patients compared to controls (FIG. 8A). Increasedcomplement activation was due to loss of CD55 as lentiviral transductionof patient T-cell blasts with CD55 protected them from complementdeposition (FIG. 8B). The finding was verified on Jurkat T cells, whichalso constitutively express cell surface CD55, after CRISPR-mediateddeletion of the CD55 gene. Increased C3d deposition was observed in thegenetically deficient (CD55-) compared to CD55-expressing (CD55+) Jurkatcells (FIG. 8C). C3b (not shown) was unable to detect, suggesting thatdeposited C3b is rapidly degraded, possibly by serum factor I and thecofactor CD46 or factor H (FIG. 6A). It is known that human intestinalepithelial cells (IECs) express CD55, and that it is upregulated uponTNFα treatment. Thus, it was hypothesized that it may protect IECs fromcomplement activation and damage by anaphylatoxin signaling or directattack of the terminal complement cascade. We therefore knocked downCD55 in the human IEC line HT29 using lentiviral shRNA transduction andfound that this permitted increased C3d deposition. Using variousconditions, it was found that the CD55 mean fluorescent intensity (MFI)was inversely correlated with C3d staining indicating that as the levelof surface CD55 decreases, the activation of complement increases (FIG.8D). In all cell types tested, lack of CD55 in patient cells led toincreased complement deposition, however, serum/complement-mediated celldeath was not observed (FIG. 8E), suggesting that CD55-deficient cellsallow early steps in complement activation to occur, but this does notprogress to terminal formation of the “membrane attack complex” (MAC).

CD55-Deficient T Cells have an Increased Inflammatory Cytokine Profile.

Complement proteins are mainly produced in the liver, but also locallyby cells of the immune system (dendritic cells and T cells). Immune cellcomplement can provide costimulatory and Thl differentiation signals toT cells through either CD46-mediated sensing of deposited C3b or throughthe C3a/C5a anaphylatoxins signaling through the anaphylatoxin receptors(AnR) C3aR and C5aR. CD55 governs complement activation and consequentimmune stimulation. Cytokine secretion by patient CD4⁺ T cells wasmeasured during primary and secondary stimulation with TCR agonisticantibodies and found increased TNFα and less IL-10 production comparedto controls (FIGS. 9A, 9B, 10A, and 10B). No consistent differences weredetected for IFNγ secretion (FIGS. 9C and 10C). To determine if thealtered cytokine secretion was dependent on increased AnR signaling,small molecule inhibitors of C3aR and C5aR1 were used to blockanaphylatoxin signaling. AnR inhibition decreased overall TNFαproduction and abrogated the difference between patient cells andcontrols (FIG. 9A). However, AnR inhibition did not rescue IL-10production in patient T cells suggesting that the IL-10 secretion defectdid not require AnR stimulation. Activated human CD4⁺ T cells expressedC3aR, but not C5aR1 or C5aR2, suggesting that C3a may play a larger rolethan C5a in T-cell activation (FIG. 9D). Interestingly, surfaceexpression of C3aR on T cells depended on the presence of all transretinoic acid (ATRA) and there was comparable expression between patientand control cells (FIG. 9E). As previously demonstrated, T cells grownin the presence of ATRA expressed the chemokine receptor CCR9 and theintegrin α4β7 that promote gut homing (FIGS. 10D and 10E). This suggeststhat gut homing T cells are also best suited to respond to anaphylatoxinproducts of complement activation.

In addition to complement regulation, CD55 mediates a co-stimulatorysignal through interactions with CD97 that enhances T-cell activationand the production of the anti-inflammatory cytokine IL-10. Patientcells failed to respond to anti-CD55 or CD97, the natural ligand forCD55, mediated costimulation as reflected by CD69 and CD25 upregulationand proliferation (FIG. 10F-10H). Additionally, patient T cells lackedCD55-dependent IL-10 production (FIGS. 9F and 101). Taken together,these data demonstrate a critical role for CD55 in controllingcomplement and non-complement mediated production of proinflammatory andanti-inflammatory cytokines, respectively.

TNFα and ATRA Regulate Complement and Coagulation Regulatory Proteins onEndothelial Cells.

CD55-deficient patients are susceptible to thrombotic events, suggestingan abnormality in the regulation of the coagulation cascade Inflammatorycytokines, such as TNFα, can modulate the coagulant and thromboticproperties of endothelial cells by down regulating thrombomodulin TM andup regulating tissue factor (TF) expression. As T cells from thepatients make more TNFα compared to controls, next it was determined howTNFα modulates complement and coagulation. As previously demonstrated,TNFα treatment induces procoagulatory TM and TF expression on thesurface of treated endothelial cells (FIGS. 9G and 9H). Interestingly,inventors also found that TNFα treatment decreased CD46 and CD59expression, while modestly increasing CD55 expression on endothelialcells (FIG. 9I-9L). It was previously determined that ATRA increasedsurface expression of C3aR on human CD4⁺ T cells and next sought todetermine if ATRA similarly affected endothelial cells. While noexpression of C3aR, C5aR, or C5aR2 was found on HUVEC cells either withor without ATRA addition (data not shown), ATRA treatment did result ina dose-dependent decrease in thrombomodulin expression with aconcomitant increase in CD55 expression and no change in either CD46 orCD59 (FIGS. 9M-9P). Combined, these data suggest that theproinflammatory cytokines preferentially secreted by patient immunecells predispose endothelial cells to the activation of both thecomplement and coagulation cascades. This may be further exacerbated byhigh local concentrations of ATRA, which are often found in intestinaltissues due to dietary intake and absorption of retinols.

Compstatin Inhibits C3d Deposition on Human Cells.

FIG. 11A illustrates that Compstatin inhibits C3d deposition on human Tcells. C3d deposition was substantially increased in patient T cellstreated with 5% nHS, as compared to control treatments and T cells. Theincrease was substantially inhibited by the addition of 100 μMCompstatin. Similarly, as shown in FIG. 11B, the ratio of C3a to C4a inculture supernatants from patient T cells after 24 hours of incubationwith 5% nHS 100 μM Compstatin was considerably decreased relative topatient samples not treated with Compstatin. In FIG. 11C, the ratio ofC5a to C4a in culture supernatants of patient T cells after 24 hours ofincubation with 5% nHS and 100 μM Compstatin is decreased, as comparedto patient T cells not treated with Compstatin. ***=p<0.001.

Discussion

A severe chronic syndrome of inherited PLE is described withlymphangiectasia, thrombotic angiopathy, and atypical IBD caused byloss-of-function (LOF) mutations in the CD55 gene. Our results show thatloss of CD55 causes dysregulated complement activation, anaphylatoxinsignaling, inflammatory cytokine production, and thrombophilia viainduction of the coagulation cascade. CD55 deficiency, Hyperactivationof complement, Angiopathic thrombosis and Protein Losing Enteropathy(CHAPLE) syndrome is defined herein as a heritable cause ofcomplement-mediated lymphangiectasia. The cohort described herein showsthat CHAPLE syndrome presents with variable expressivity, fromsubclinical to life threatening presentations, potentially explainingthe variation in intestinal disease previously associated with the Inabphenotype.

Complement is a tightly regulated system of plasma and cell surfaceproteins that promotes the phagocytosis and destruction of microbes andmodulates the local immune response through soluble anaphylatoxins.Inherited or acquired defects in complement inhibitory proteins lead todistinct syndromes in different organ systems which include paroxysmalnocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome(aHUS), dense deposit disease, and age-related macular degeneration(FIG. 6A). PNH results from somatic mutations of the PIGA gene inhematopoietic precursors. This leads to the loss of GPI-anchoredcomplement inhibitory proteins, including CD55 and CD59, causingterminal-complement mediated hemolysis with secondary thrombotic riskdue to platelet activation. Germline LOF mutations affecting thecomplement regulatory proteins Factor H, Factor I, and CD46 or theanti-coagulatory protein thrombomodulin, result in the development ofaHUS which is characterized by activation and damage of glomerularmicrovascular endothelial cells by complement intermediates and themembrane-attack complex, hemolysis due to mechanical injury, and kidneyfailure due to microthrombotic angiopathy. Defects in these same genescan cause age-related macular degeneration due to complement depositionin the retina. Unlike PNH and aHUS, hemolysis or kidney failure was notobserved in CHAPLE patients. By contrast, CHAPLE patients, like PNH andaHUS patients, have an increased risk of thrombosis, highlighting thatexcessive complement activation can lead to induction of the coagulationcascade. The differences in clinical phenotype between PNH, aHUS, andCHAPLE syndrome may be due to varying cellular tropisms of the affectedcomplement regulatory proteins or due to differences in the extent ofcomplement activation. For instance, hemolysis or complement-dependentcell death was not detected in CHAPLE patients, suggesting little, ifany, membrane-attack complex forms, differentiating this disease fromPNH and aHUS.

Interestingly, it was observed that CD55 deficiency primarily manifestsas an intestinal disease, occasionally complicated by life-threateningthromboembolism. The locations affected by thrombotic disease in CHAPLEpatients (abdomen and brain) was similar to PNH and dissimilar to aHUS,which primarily affects the kidneys. This may reflect expressionpatterns of CD55 and other complement regulatory proteins. Supportingthis idea, CD55 protein expression was found to be increased onendothelial cells in response to both TNFα and ATRA. During normalimmune responses TNFα induction of CD55 may limit complement activationand prevent host damage. ATRA is a biologically active metabolite ofVitamin A/retinol, which is strictly acquired through diet. As such,intestinal tissues are exposed to a high amount of retinol and itsmetabolites and these metabolites play key roles in controlling theintestinal innate and adaptive immune response. Another possibility isthat the constant microbial challenge in the intestinal tractpredisposes this area to complement activation and activation of innateand adaptive immune responses, which may be hyperactivated by theabsence of CD55. Additionally, TNFα and IL-10, whose production isaltered in patient T cells, are critical for the regulation of universalinflammatory responses and deficiency of these cytokines preferentiallycauses intestinal disease, suggesting that intestinal tissues aresensitive to changes in the inflammatory cytokine milieu. Given theobservation that ATRA drives both C3aR expression and a gut homingphenotype on human CD4⁺ T cells, it is likely that T cells in theintestinal tissues are sensitized to the detection and activation bycomplement components. Collectively, these factors likely drive theintestinal disease associated with CD55 deficiency.

The hallmarks of CHAPLE syndrome or disease are early-onset PLE withlymphangiectasia and IBD. This is attributable to 3 relevant pathogenicmechanisms: i) impaired endothelial or epithelial barrier function dueto signaling by anaphylatoxins or sub-lytic MAC deposition, ii)increased pressure due to thrombotic events, and iii) changes to theinflammatory cytokine environment. CD55-deficient mice show increasedcomplement-dependent susceptibility to dextran sodium sulfate(DSS)-induced colitis, with complement deposition on IECs. Thethrombotic events in CHAPLE patients, when localized to mesenterictissue, may drive increased lymphatic pressure and exacerbate intestinallymphangiectasia and chronic PLE. CD55-deficient patient T cells producemore TNFα and less IL-10, thereby creating a pro-inflammatory milieu.TNFα production was found to be dependent on anaphylatoxin signaling,while decreased IL-10 production was likely due to loss of CD55-mediatedT-cell costimulation. CD55-deficient mice show similar alterations incytokines. Taken together, these data show that complement activation iscritical to disease pathogenesis (FIGS. 12A and 12B).

The patients with CHAPLE disease described herein have a chronic andseverely debilitating, sometimes fatal, clinical course. As suchpatients with early-onset PLE should be screened for CD55 expression.CHAPLE patients have responded well to protein infusion and resection oflocalized lymphangiectatic intestinal segments; though albumin infusionprovides only a transient palliation of symptoms and patients requireongoing treatment. Attempts to treat this disorder with conventionalimmunosuppressants, somatostatin analogues, or anticoagulants have beenmet with only a moderate degree of success in individual patients. Giventhe complement-mediated pathogenesis of this disorder, it is believedthat agents that inhibit the complement-pathway (e.g., thecomplement-inhibitory monoclonal antibody Eculizumab, which has beenused successfully in PNH and aHUS, or Compstatin), would be effective attreating CHAPLE patients. Eculizumab blocks C5 cleavage, therebypreventing the formation of the potent anaphylatoxin C5a and progressionto the terminal complement cascade, two critical mediators of effectorfunction in the complement cascade. While CD55 regulates the C5convertase, making Eculizumab a good target therapy, it also regulatesthe C3 convertase. Furthermore, Compstatin is a peptide inhibitor (i.e.,a cyclic tridecapeptide) of the complement system. In particular,Compstatin binds component C3, thereby inhibiting C3activation/cleavage. As such, any complement-pathway inhibitors,including C3 convertase and C5 convertase inhibitors are possibletherapeutic agents for treating patients with CHAPLE disease.

Specific Embodiments

An aspect provided by the present disclosure is a method of diagnosing apatient with CD55 deficiency, hyperactivation of complement, angiopathicthrombosis and protein losing enteropathy (CHAPLE) is provided. Themethod comprises: providing a sample from a patient; detecting at leastone of: at least one mutation in a DNA sequence of a CD55 gene; at leastone mutation in a RNA sequence of a CD55 transcript; at least onemutation in a DNA sequence of a CD55 complementary DNA (cDNA); CD55protein; CD55 protein binding; complement deposition; or combinationsthereof, wherein a mutation in the CD55 gene, transcript or cDNA, orabsence or decrease in activity of CD55 protein is indicative of apatient at risk of developing or having CHAPLE; and diagnosing thepatient as having or not having CHAPLE.

In any aspect or embodiment described herein, the patient is diagnosedwith CHAPLE when at least one of the following is detected: at least onemutation in a DNA sequence, an RNA sequence or a cDNA sequence of CD55that results in a CD55 protein with substantially diminished functionalactivity, a CD55 protein with no functional activity, a lack ofexpression of CD55 protein (i.e., no CD55 protein expression), or asubstantially diminished expression of CD55 protein; a CD55 protein withsubstantially diminished functional activity; a CD55 protein with nofunctional activity; a lack of expression of CD55 protein (i.e., no CD55protein expression); a substantially diminished expression of CD55protein; or combinations thereof.

In any aspect or embodiment described herein, the patient is diagnosedwith CHAPLE when the patient has at least one CHAPLE related symptom andat least one of the following is detected: at least one mutation in aDNA sequence, an RNA sequence or a cDNA sequence of CD55 that results ina CD55 protein with substantially diminished functional activity, a CD55protein with no functional activity, a lack of expression of CD55protein (i.e., no CD55 protein expression), or a substantiallydiminished expression of CD55 protein; a CD55 protein with substantiallydiminished functional activity; a CD55 protein with no functionalactivity; a lack of expression of CD55 protein (i.e., no CD55 proteinexpression); a substantially diminished expression of CD55 protein; orcombinations thereof.

In any aspect or embodiment described herein, the CHAPLE related symptomis selected from the group consisting of: inflammatory bowel disease,enteropathy, protein losing enteropathy, protein losing enteropathyassociated with hypoalbuminemia, hypoalbuminemia, hypogammaglobulinemia,intestinal lymphangiectasia, lymphangiectasia, thrombotic events,thromboembolism, hyperactivation of complement, angiopathic thrombosis,hypoproteinemia, or combinations thereof.

In any aspect or embodiment described herein, the method furthercomprises administering an effective amount of a composition comprisingat least one complement inhibitor to the subject with CHAPLE, whereinthe composition is effective in treating or preventing at least onesymptom of CHAPLE.

In any aspect or embodiment described herein, the complement inhibitoris selected from the group consisting of a serine protease inhibitor, asoluble complement regulator, a therapeutic antibody or anantigen-binding fragment thereof, a complement component inhibitor, andan anaphylatoxin receptor antagonist.

In any aspect or embodiment described herein, the mutation in the DNAsequence of the CD55 gene or in the RNA sequence of the CD55 transcriptresults in a near to complete absence of CD55 protein expression or theexpression of CD55 protein with substantially diminished function orthat is non-functional.

In any aspect or embodiment described herein, the mutation in the DNAsequence of the CD55 gene is at least one of: c.149-150delAA;c.149-150insCCTT; c.109delC; c.800G>C; c.287-1G>C;c.149-150delAAinsCCTT; or combinations thereof.

In any aspect or embodiment described herein, detecting includes atleast one of: sequencing at least a portion of the CD55 gene or the CD55transcript or the CD55 cDNA; or contacting a labeled nucleic acid probeto at least a portion of the CD55 gene or the CD55 transcript or theCD55 cDNA; or contacting at least a portion of the CD55 gene or CD55transcripts or cDNA thereof with a microarray; or combinations thereof.

In any aspect or embodiment described herein, hybridization of thelabeled nucleic acid probe is indicative of a mutation in the portion ofthe CD55 gene or CD55 transcript.

In any aspect or embodiment described herein, hybridization of thelabeled nucleic acid probe is indicative of the portion of the CD55 geneor CD55 transcript having a wild-type sequence at the location ofhybridization.

In any aspect or embodiment described herein, sequencing at least aportion of the CD55 gene or CD55 transcript or cDNA thereof includesamplifying at least one region of interest for sequencing with at leastone polymerase chain reaction (PCR) with at least one of the followingprimer sets: CTACTCCACCCGTCTTGTTTGT and TTTGGGGGTTAAGGATACAGTC (Exon 1);CAGGTGTGGCATTTCAAGG and ACCCTGGGGTTTAGTAACGC (Exon 2);AAGTACTAAATATGCGCAAAGCAG and ATGGTCCTATCAAGAAACATCC (Exon 3);GTTACCTTCTTTGTGTGTATGCC and GCTGTGAATACCAGTCATGAAAC (Exon 4);AACCTGGAGAATTTGAGGAAAG and TGTGCTAATATTCTTAAGGGGC (Exon 5);GCATTTATAAGCATCTCTTGTTGG and TCATTGAATGTCTGCAACCC (Exon 6);CTAGGTGTTTGTGGGGAGAGAG and TCTGGTGGGTTTCTGAAGAGTT (Exon 7);TTTACGCAGAGTCCTTCAGC and CCATTTAATCCTGCAATCTTGG (Exon 8);TGGAAATTTGAGTTGCTTTCG and TCTCCCAGGAATATGGATTG (Exon 9);GCACCCCAAATTAACTGATTC and ATGTGATTCCAGGACTGCC (Exon 10); or combinationsthereof.

In any aspect or embodiment described herein, contacting a labelednucleic acid probe to at least a portion of the CD55 gene or the CD55transcript or the CD55 cDNA is performed using real-time PCR.

In any aspect or embodiment described herein, contacting a labelednucleic acid probe to at least a portion of the CD55 transcripts or cDNAthereof comprises: isolating CD55 transcripts; reverse transcribing atleast a portion of the CD55 transcripts; and contacting the cDNA withthe labeled nucleic acid probe.

In any aspect or embodiment described herein, the microarray includesprobes designed to detect DNA, transcript, or cDNA mutations that resultin the complete absence in CD55 protein or a non-functional CD55protein.

In any aspect or embodiment described herein, detecting CD55 proteincomprises: contacting the sample with at least one CD55 bindingpolypeptide.

In any aspect or embodiment described herein, the CD55 bindingpolypeptide includes a detectable label.

In any aspect or embodiment described herein, the binding polypeptide isan anti-CD55 antibody or a CD55-binding fragment thereof.

In any aspect or embodiment described herein, detecting CD55 proteinfurther comprises contacting the sample or a CD55-CD55 bindingpolypeptide complex with at least one secondary polypeptide that bindsspecifically to the CD55 binding polypeptide.

In any aspect or embodiment described herein, the secondary polypeptideincludes a detectable label.

In any aspect or embodiment described herein, the secondary polypeptideis an antibody or fragment thereof that binds the CD55 bindingpolypeptide.

In any aspect or embodiment described herein, the detecting of CD55protein is performed using at least one of a western blot, flowcytometry, an immunoassay, or combinations thereof.

In any aspect or embodiment described herein, the immunoassay is atleast one assay selected from the group consisting of enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay, magnetic immunoassay,enzyme-linked immunospot (ELISPOT), immunofluorescence; and combinationsthereof.

In any aspect or embodiment described herein, the detecting of CD55binding function includes examining the at least one of C3b affinity,C3b avidity, C4b affinity, C4b avidity, or combinations thereof.

In any aspect or embodiment described herein, detecting complementdeposition includes detecting C3d deposition.

A further aspect provided by the present disclosure is a method oftreating a patient with CD55 deficiency, hyperactivation of complement,angiopathic thrombosis and protein losing enteropathy (CHAPLE) orpreventing at least one symptom of CHAPLE in a patient at risk ofdeveloping the same. The method comprises: administering an effectiveamount of a composition comprising at least one complement inhibitor toa subject in need thereof, wherein the composition is effective intreating or preventing at least one symptom of CHAPLE.

In any aspect or embodiment described herein, the complement inhibitoris selected from the group consisting of a serine protease inhibitor, asoluble complement regulator, a therapeutic antibody or anantigen-binding fragment thereof, a complement component inhibitor, andan anaphylatoxin receptor antagonist.

In any aspect or embodiment described herein, the serine proteaseinhibitors is at least one of a C3 convertase inhibitor, a C5 convertaseinhibitor, a C1 inhibitor, a C1r inhibitor, a C1s inhibitor, a C2ainhibitor, a MASP-1 inhibitor, a MASP-2 inhibitor, a factor D inhibitor,a factor B inhibitor, a factor I inhibitor or combinations thereof.

In any aspect or embodiment described herein, the serine proteaseinhibitor is at least one of BCX-1470 (BioCryst, Birmingham, Ala., USA),C1s-INH-248 (Knoll/Abbott, Abbott Park, Ill., USA), compstatin, Cetor®(Sanquin, Amsterdam, Netherlands), Berinert® (CSL Behring, King ofPrussia, Pa., USA), Cinryze™ (ViroPharma, Exton, Pa., USA), rhC 1INH

(Pharming Group N.V., Leiden, Netherlands), Ruconest® (SalixPharmaceuticals, Inc., Raleigh, N.C., USA) or combinations thereof.

In any aspect or embodiment described herein, the soluble complementregulator is at least one of a soluble form of a membrane cofactorprotein (MCP or CD46), a soluble form of a decay-accelerating factor(DAF or CD55), a soluble form of a membrane attack complex-inhibitorprotein (MAC-IP or CD59), a soluble form of complement receptor 1 (CD35)or combinations thereof.

In any aspect or embodiment described herein, the soluble complementregulator is at least one of sCR1 (TP10; Advant Immunotherapeutics,Needham, Mass., USA), sCR1-sL^(ex) (TP10; Advant Immunotherapeutics,Needham, Mass., USA), sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge,Mass., USA), a membrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or combinationsthereof.

In any aspect or embodiment described herein, the therapeutic antibodyor the antigen-binding fragment thereof is at least one polypeptide thatbinds C3, C3a, C3b, C3 convertase, C5, C5a, C5b, C5 convertase, C7, C8,or C9, factor B, factor D, C4, C2, C1, properdin, a functional blockingantibody of an anaphylatoxin or combinations thereof, wherein saidbinding inhibits complement activation by at least one of blockingassociation/binding with other complement proteins, blockingassociation/binding with receptor proteins, blocking serine proteaseactivity or combinations thereof.

In any aspect or embodiment described herein, the therapeutic antibodyor the antigen-binding fragment thereof is at least one of eculizumab(Soliris®; Alexion Pharmaceuticals Inc., New Haven, Conn., USA),ALXN1007 (Alexion Pharmaceuticals Inc., New Haven, Conn., USA),neutrazumab (G2 Therapies, Darlinghurst, NSW, Australia), Pexelizumab(Alexion Pharmaceuticals Inc., New Haven, Conn., USA), ofatumumab(Genmab A/S, Copenhagen, Denmark), HuMax-CD38 (Benmab A/S, Copenhagen,Denmark), TNX-558 (Tanox, South San Francisco, Calif., USA), TNX-234(Tanox, South San Francisco, Calif., USA), TA106 (Taligen, Aurora,Colo., USA), anti-properdin (Novelmed, Cleveland, Ohio, USA) orcombinations thereof.

In any aspect or embodiment described herein, the complement componentinhibitor is a peptide, nucleic acids, a synthetic molecule or acomination thereof that disrupts protein functions by steric hindranceor the induction of conformational changes.

In any aspect or embodiment described herein, the complement componentinhibitor is at least one of compstatin, anti-C5 RNA aptamer (ARC1905;Archemix, Cambridge, Mass., USA), or analogs or derivatives thereof, orcombinations thereof.

In any aspect or embodiment described herein, the anaphylatoxin receptorantagonist is at least one of a C5aR antagonist, a C5L2 antagonist, aC3a receptor antagonist, a functional blocking antibody of ananaphylatoxin or combinations thereof.

In any aspect or embodiment described herein, the anaphylatoxin receptorantagonist is at least one of PMX-53 (PepTech Corp, Bedform, Mass.,USA), PMX-205 (PepTech Corp, Bedform, Mass., USA), JPE-1375 (Jerini,Berlin, Germany), JSM-7717 (Jerini, Berlin, Germany), rhMBL (EnzonPharmaceuticals, Cranford, N.J., USA) or combinations thereof.

An additional aspect provided by the present disclosure is a therapeuticcomposition for treating or preventing at least one symptom of CD55deficiency, hyperactivation of complement, angiopathic thrombosis andprotein losing enteropathy (CHAPLE). The composition comprises: aneffective amount of two or more agents, wherein at least one of theagents is a complement inhibitor to a subject in need thereof; and apharmaceutically acceptable carrier, wherein the composition iseffective in treating or preventing at least one symptom of CHAPLE.

In any aspect or embodiment described herein, the complement inhibitoris selected from the group consisting of a serine protease inhibitor, asoluble complement regulator, a therapeutic antibody or anantigen-binding fragment thereof, a complement component inhibitor, andan anaphylatoxin receptor antagonist.

In any aspect or embodiment described herein, the complement inhibitorsinclude a C3 convertase inhibitor and a C5 convertase inhibitor.

In any aspect or embodiment described herein, the serine proteaseinhibitors is at least one of a C3 convertase inhibitor, a C5 convertaseinhibitor, a C1 inhibitor, a C1r inhibitor, a C1s inhibitor, a C2ainhibitor, a MASP-1 inhibitor, a MASP-2 inhibitor, a factor D inhibitor,a factor B inhibitor, a factor I inhibitor or combinations thereof.

In any aspect or embodiment described herein, the serine proteaseinhibitor is at least one of BCX-1470 (BioCryst, Birmingham, Ala., USA),C1s-INH-248 (Knoll/Abbott, Abbott Park, Ill., USA), compstatin, Cetor®(Sanquin, Amsterdam, Netherlands), Berinert® (CSL Behring, King ofPrussia, Pa., USA), Cinryze™ (ViroPharma, Exton, Pa., USA), rhC 1INH(Pharming Group N.V., Leiden, Netherlands), Ruconest® (SalixPharmaceuticals, Inc., Raleigh, N.C., USA) or combinations thereof.

In any aspect or embodiment described herein, the soluble complementregulator is at least one of a soluble form of a membrane cofactorprotein (MCP or CD46), a soluble form of a decay-accelerating factor(DAF or CD55), a soluble form of a membrane attack complex-inhibitorprotein (MAC-IP or CD59), a soluble form of complement receptor 1 (CD35)or combinations thereof.

In any aspect or embodiment described herein, the soluble complementregulator is at least one of sCR1 (TP10; Advant Immunotherapeutics,Needham, Mass., USA), sCR1-sL^(ex) (TP10; Advant Immunotherapeutics,Needham, Mass., USA), sDAG-sMCP hybrid (MLN-2222, Millennium, Cambridge,Mass., USA), a membrane-tethered sCD59 (Mirococept or APT070; InflazymePharmaceuticals, Vancouver, British Columbia, Canada) or combinationsthereof.

In any aspect or embodiment described herein, the therapeutic antibodyor the antigen-binding fragment thereof is at least one polypeptide thatbinds C3, C3a, C3b, C3 convertase, C5, C5a, C5b, C5 convertase, C7, C8,or C9, factor B, factor D, C4, C2, C1, properdin, a functional blockingantibody of an anaphylatoxin or combinations thereof, wherein saidbinding inhibits complement activation by at least one of blockingassociation/binding with other complement proteins, blockingassociation/binding with receptor proteins, blocking serine proteaseactivity or combinations thereof.

In any aspect or embodiment described herein, the therapeutic antibodyor the antigen-binding fragment thereof is at least one of eculizumab(Soliris®; Alexion Pharmaceuticals Inc., New Haven, Conn., USA),ALXN1007 (Alexion Pharmaceuticals Inc., New Haven, Conn., USA),neutrazumab (G2 Therapies, Darlinghurst, NSW, Australia), Pexelizumab(Alexion Pharmaceuticals Inc., New Haven, Conn., USA), ofatumumab(Genmab A/S, Copenhagen, Denmark), HuMax-CD38 (Benmab A/S, Copenhagen,Denmark), TNX-558 (Tanox, South San Francisco, Calif., USA), TNX-234(Tanox, South San Francisco, Calif., USA), TA106 (Taligen, Aurora,Colo., USA), anti-properdin (Novelmed, Cleveland, Ohio, USA) orcombinations thereof.

In any aspect or embodiment described herein, the complement componentinhibitor is a peptide, nucleic acids, a synthetic molecule or acombination thereof that disrupts protein functions by steric hindranceor the induction of conformational changes.

In any aspect or embodiment described herein, the complement componentinhibitor is at least one of compstatin, anti-C5 RNA aptamer (ARC1905;Archemix, Cambridge, Mass., USA), or analogs or derivatives thereof, orcombinations thereof.

In any aspect or embodiment described herein, the anaphylatoxin receptorantagonist is at least one of a C5aR antagonist, a C5L2 antagonist, aC3a receptor antagonist, a functional blocking antibody of ananaphylatoxin or combinations thereof.

In any aspect or embodiment described herein, the anaphylatoxin receptorantagonist is at least one of PMX-53 (PepTech Corp, Bedform, Mass.,USA), PMX-205 (PepTech Corp, Bedform, Mass., USA), JPE-1375 (Jerini,Berlin, Germany), JSM-7717 (Jerini, Berlin, Germany), rhMBL (EnzonPharmaceuticals, Cranford, N.J., USA) or combinations thereof.

It is understood that the detailed examples and embodiments describedherein are given by way of example for illustrative purposes only, andare in no way considered to be limiting to the invention. Variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are included within the spirit and purview ofthis application and are considered within the scope of the appendedclaims. For example, the relative quantities of the ingredients may bevaried to optimize the desired effects, additional ingredients may beadded, and/or similar ingredients may be substituted for one or more ofthe ingredients described. Additional advantageous features andfunctionalities associated with the systems, methods, and processes ofthe present invention will be apparent from the appended claims.

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1. A method of diagnosing a patient with CD55 deficiency,hyperactivation of complement, angiopathic thrombosis and protein losingenteropathy (CHAPLE), the method comprising: providing a sample from apatient; detecting at least one of: at least one mutation in a DNAsequence of a CD55 gene; at least one mutation in a RNA sequence of aCD55 transcript; at least one mutation in a DNA sequence of a CD55complementary DNA (cDNA); CD55 protein; CD55 protein binding; complementdeposition; or combinations thereof, wherein a mutation in the CD55gene, transcript or cDNA, or absence or decrease in activity of CD55protein is indicative of a patient at risk of developing or havingCHAPLE; and diagnosing the patient as having or not having CHAPLE. 2.The method of claim 1, wherein the patient is diagnosed with CHAPLE whenat least one of the following is detected: at least one mutation in aDNA sequence, an RNA sequence or a cDNA sequence of CD55 that results ina CD55 protein with substantially diminished functional activity, a CD55protein with no functional activity, a lack of expression of CD55protein (i.e., no CD55 protein expression), or a substantiallydiminished expression of CD55 protein; a CD55 protein with substantiallydiminished functional activity; a CD55 protein with no functionalactivity; a lack of expression of CD55 protein (i.e., no CD55 proteinexpression); a substantially diminished expression of CD55 protein; orcombinations thereof.
 3. The method of claim 1, wherein the patient isdiagnosed with CHAPLE when the patient has at least one CHAPLE relatedsymptom and at least one of the following is detected: at least onemutation in a DNA sequence, an RNA sequence or a cDNA sequence of CD55that results in a CD55 protein with substantially diminished functionalactivity, a CD55 protein with no functional activity, a lack ofexpression of CD55 protein (i.e., no CD55 protein expression), or asubstantially diminished expression of CD55 protein; a CD55 protein withsubstantially diminished functional activity; a CD55 protein with nofunctional activity; a lack of expression of CD55 protein (i.e., no CD55protein expression); a substantially diminished expression of CD55protein; or combinations thereof.
 4. The method of claim 3, wherein theCHAPLE related symptom is selected from the group consisting of:inflammatory bowel disease, enteropathy, protein losing enteropathy,protein losing enteropathy associated with hypoalbuminemia,hypoalbuminemia, hypogammaglobulinemia, intestinal lymphangiectasia,lymphangiectasia, thrombotic events, thromboembolism, hyperactivation ofcomplement, angiopathic thrombosis, hypoproteinemia, or combinationsthereof.
 5. The method of claim 1, further comprising administering aneffective amount of a composition comprising at least one complementinhibitor to the subject with CHAPLE, wherein the composition iseffective in treating or preventing at least one symptom of CHAPLE,wherein the complement inhibitor is selected from the group consistingof a serine protease inhibitor, a soluble complement regulator, atherapeutic antibody or an antigen-binding fragment thereof, acomplement component inhibitor, and an anaphylatoxin receptorantagonist.
 6. (canceled)
 7. The method of claim 1, wherein the mutationin the DNA sequence of the CD55 gene or in the RNA sequence of the CD55transcript results in a near to complete absence of CD55 proteinexpression or the expression of CD55 protein with substantiallydiminished function or that is non-functional.
 8. The method of claim 1,wherein the mutation in the DNA sequence of the CD55 gene is at leastone of: c.149-150delAA; c.149-150insCCTT; c.109delC; c.800G>C;c.287-1G>C; c.149-150delAAinsCCTT; or combinations thereof.
 9. Themethod of claim 1, wherein detecting includes at least one of:sequencing at least a portion of the CD55 gene or the CD55 transcript orthe CD55 cDNA; or contacting a labeled nucleic acid probe to at least aportion of the CD55 gene or the CD55 transcript or the CD55 cDNA; orcontacting at least a portion of the CD55 gene or CD55 transcripts orcDNA thereof with a microarray; or combinations thereof.
 10. The methodof claim 9, wherein hybridization of the labeled nucleic acid probe isindicative of a mutation in the portion of the CD55 gene or CD55transcript.
 11. The method of claim 9, wherein hybridization of thelabeled nucleic acid probe is indicative of the portion of the CD55 geneor CD55 transcript having a wild-type sequence at the location ofhybridization.
 12. The method of claim 9, wherein sequencing at least aportion of the CD55 gene or CD55 transcript or cDNA thereof includesamplifying at least one region of interest for sequencing with at leastone polymerase chain reaction (PCR) with at least one of the followingprimer sets: (Exon 1) CTACTCCACCCGTCTTGTTTGT and TTTGGGGGTTAAGGATACAGTC;(Exon 2) CAGGTGTGGCATTTCAAGG and ACCCTGGGGTTTAGTAACGC; (Exon 3)AAGTACTAAATATGCGCAAAGCAG and ATGGTCCTATCAAGAAACATCC; (Exon 4)GTTACCTTCTTTGTGTGTATGCC and GCTGTGAATACCAGTCATGAAAC; (Exon 5)AACCTGGAGAATTTGAGGAAAG and TGTGCTAATATTCTTAAGGGGC; (Exon 6)GCATTTATAAGCATCTCTTGTTGG and TCATTGAATGTCTGCAACCC; (Exon 7)CTAGGTGTTTGTGGGGAGAGAG and TCTGGTGGGTTTCTGAAGAGTT; (Exon 8)TTTACGCAGAGTCCTTCAGC and CCATTTAATCCTGCAATCTTGG; (Exon 9)TGGAAATTTGAGTTGCTTTCG and TCTCCCAGGAATATGGATTG; (Exon 10) GCACCCCAAATTAACTGATTC and ATGTGATTCCAGGACTGCC; or combinations thereof.


13. The method of claim 9, wherein contacting a labeled nucleic acidprobe to at least a portion of the CD55 gene or the CD55 transcript orthe CD55 cDNA is performed using real-time PCR.
 14. The method of claim9, wherein contacting a labeled nucleic acid probe to at least a portionof the CD55 transcripts or cDNA thereof comprises: isolating CD55transcripts; reverse transcribing at least a portion of the CD55transcripts; and contacting the cDNA with the labeled nucleic acidprobe.
 15. The method of claim 1, wherein the microarray includes probesdesigned to detect DNA, transcript, or cDNA mutations that result in thecomplete absence in CD55 protein or a non-functional CD55 protein. 16.The method of claim 9, wherein detecting CD55 protein comprises:contacting the sample with at least one CD55 binding polypeptide,wherein the binding peptide includes a detectable label, or is ananti-CD55 antibody or a CD55-binding fragment of an antibody. 17.-23.(canceled)
 24. The method of claim 1, wherein the detecting of CD55binding function includes examining the at least one of C3b affinity,C3b avidity, C4b affinity, C4b avidity, or combinations thereof.
 25. Themethod of claim 1, wherein detecting complement deposition includesdetecting C3d deposition.
 26. A method of treating a patient having CD55deficiency, hyperactivation of complement, angiopathic thrombosis andprotein losing enteropathy (CHAPLE) or preventing at least one symptomof CHAPLE in a patient at risk of developing the same, the methodcomprising: administering an effective amount of a compositioncomprising at least one complement inhibitor to a subject in needthereof, wherein the composition is effective in treating or preventingat least one symptom of CHAPLE.
 27. The method of claim 26, wherein thecomplement inhibitor is selected from the group consisting of a serineprotease inhibitor, a soluble complement regulator, a therapeuticantibody or an antigen-binding fragment thereof, a complement componentinhibitor, and an anaphylatoxin receptor antagonist.
 28. The method ofclaim 27, wherein: the serine protease inhibitor is at least one of a C3convertase inhibitor, a C5 convertase inhibitor, a C1 inhibitor, a C1rinhibitor, a C1s inhibitor, a C2a inhibitor, a MASP-1 inhibitor, aMASP-2 inhibitor, a factor D inhibitor, a factor B inhibitor, a factor Iinhibitor or combinations thereof; the soluble complement regulator isat least one of a soluble form of a membrane cofactor protein (MCP orCD46), a soluble form of a decay-accelerating factor (DAF or CD55), asoluble form of a membrane attack complex-inhibitor protein (MAC-IP orCD59), a soluble form of complement receptor 1 (CD35) or combinationsthereof; the therapeutic antibody or the antigen-binding fragmentthereof is at least one polypeptide that binds C3, C3a, C3b, C3convertase, C5, C5a, C5b, C5 convertase, C7, C8, or C9, factor B, factorD, C4, C2, C1, properdin, a functional blocking antibody of ananaphylatoxin or combinations thereof, wherein said binding inhibitscomplement activation by at least one of blocking association/bindingwith other complement proteins, blocking association/binding withreceptor proteins, blocking serine protease activity or combinationsthereof; the complement component inhibitor is a peptide, nucleic acids,a synthetic molecule or a combination thereof that disrupts proteinfunctions by steric hindrance or the induction of conformationalchanges; and the anaphylatoxin receptor antagonist is at least one of aC5aR antagonist, a C5L2 antagonist, a C3a receptor antagonist, afunctional blocking antibody of an anaphylatoxin or combinationsthereof. 29.-37. (canceled)
 38. A therapeutic composition for treatingor preventing at least one symptom of CD55 deficiency, hyperactivationof complement, angiopathic thrombosis and protein losing enteropathy(CHAPLE), the composition comprising: an effective amount of two or moreagents, wherein at least one of the agents is a complement inhibitor toa subject in need thereof; and a pharmaceutically acceptable carrier,wherein the composition is effective in treating or preventing at leastone symptom of CHAPLE.
 39. The composition of claim 38, wherein thecomplement inhibitor is selected from the group consisting of a serineprotease inhibitor, a soluble complement regulator, a therapeuticantibody or an antigen-binding fragment thereof, a complement componentinhibitor, and an anaphylatoxin receptor antagonist.
 40. (canceled) 41.The composition of claim 39, wherein: the serine protease inhibitors isat least one of a C3 convertase inhibitor, a C5 convertase inhibitor, aC1 inhibitor, a C1r inhibitor, a C1s inhibitor, a C2a inhibitor, aMASP-1 inhibitor, a MASP-2 inhibitor, a factor D inhibitor, a factor Binhibitor, a factor I inhibitor or combinations thereof; the solublecomplement regulator is at least one of a soluble form of a membranecofactor protein (MCP or CD46), a soluble form of a decay-acceleratingfactor (DAF or CD55), a soluble form of a membrane attackcomplex-inhibitor protein (MAC-IP or CD59), a soluble form of complementreceptor 1 (CD35) or combinations thereof; the therapeutic antibody orthe antigen-binding fragment thereof is at least one polypeptide thatbinds C3, C3a, C3b, C3 convertase, C5, C5a, C5b, C5 convertase, C7, C8,or C9, factor B, factor D, C4, C2, C1, properdin, a functional blockingantibody of an anaphylatoxin or combinations thereof, wherein saidbinding inhibits complement activation by at least one of blockingassociation/binding with other complement proteins, blockingassociation/binding with receptor proteins, blocking serine proteaseactivity or combinations thereof; the complement component inhibitor isa peptide, nucleic acids, a synthetic molecule or a combination thereofthat disrupts protein functions by steric hindrance or the induction ofconformational changes; and the anaphylatoxin receptor antagonist is atleast one of a C5aR antagonist, a C5L2 antagonist, a C3a receptorantagonist, a functional blocking antibody of an anaphylatoxin orcombinations thereof. 42.-50. (canceled)